Methods and compositions involving endopeptidases PepO2 and PepO3

ABSTRACT

The present invention concerns the methods and compositions involving endopeptidase enzymes, especially PepO2 and PepO3 from  L. helveticus , and their use in reducing bitterness by cleaving bitter peptides. In particular embodiments of the invention, these methods and compositions apply to the cheesemaking process. The invention also concerns the use of PepO2 and/or PepO3 polypeptides in the treatment or prevention of celiac sprue or as a food additive.

BACKGROUND OF THE INVENTION

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 60/480,536 filed Jun. 20, 2003, which ishereby incorporated by reference in its entirety.

1. Field of the Invention

This invention was made with government support under grant number99-CRHF-0-6055 awarded by the United States Department ofAgriculture/CREES. The government has certain rights in the invention.

The present invention relates generally to the fields of microbiologyand enzymology. More particularly, it concerns methods and compositionsinvolving endopeptidase enzymes from bacteria that cleave peptides,particularly bitter peptides and peptides involved in gluteninflammation. In some embodiments, methods and compositions concernPepO2 and/or PepO3, and their use in reducing bitterness in foods, suchas cheese, or treating or preventing celiac sprue.

2. Description of Related Art

Lactobacillus helveticus belongs to a group of organisms known as lacticacid bacteria (LAB), which are defined by the production of lactic acidas a major product of carbohydrate fermentation. Lb. helveticus hasmultiple amino acid (AA) auxotrophies and thus is dependent on transportof AA and/or transport and hydrolysis of exogenous peptides to satisfythese nutritional requirements. In AA defined media, Lb. helveticusCNRZ32 can grow without Ala, Asn, Cys, Gln, Gly, and Ser when they areabsent individually (Christensen, 2000). The fermentation of Bos taurusmilk is a common system to study the proteolytic system and physiologyof Lb. helveticus, providing a relatively consistent environment andwell characterized set of proteins as a starting point, as well ashaving adaptive significance for dairy related LAB. Since all of theidentified peptidases of L. helveticus are believed to be intracellular,the acquisition of AA is also likely to be dependent on the activity ofat least one extracellular proteinase capable of hydrolyzing caseinsinto transportable peptides (Kunji et al., 1996). Therefore, obtainingAA through the hydrolysis of caseins (the preferentially hydrolyzed milkproteins) requires a complex proteolytic system comprised ofproteinase(s), endopeptidase(s), aminopeptidase(s), tripeptidase(s),dipeptidase(s), and peptide transport systems (Christensen et al., 1999;Kunji et al., 1996; Pritchard and Coolbear, 1993).

The proteolytic systems of dairy LAB have received extensive researchattention due to their importance in the physiology of these organismsand cheese flavor development. Because LAB are fastidious microorganismswith multiple amino acid auxotrophies (Kok and De Vos, 1994), duringgrowth in milk, LAB rely on their proteolytic systems to obtainessential amino acids from caseins (CNs), the most abundant proteins inmilk (Christensen et al., 1999; Kunji et al., 1996). In many cheesevarieties, enzymatic conversion of large, casein (CN)-derived peptidesinto small peptides and free amino acids by LAB has pronounced effectson cheese flavor development as well as cheese functional properties.Additionally, proteolytic enzymes from LAB produce flavor compounds andprecursors that are essential for cheese flavor development (Christensenet al, 1999; Mulholland, 1997).

Proteolytic systems of LAB can be functionally divided into threecomponents: (i) cell envelope-associated proteinases which hydrolyzecaseins to oligopeptides; (ii) peptide transport systems, of which theoligopeptide transport system is of greatest importance in milk andcheese; (iii) and numerous intracellular peptidases (Christensen et al.,1999; Kunji et al., 1996). The intracellular peptidases of LAB consistof both endopeptidases and aminopeptidases. Endopeptidases, due to theirability to hydrolyze peptide bonds within a peptide, are of particularinterest in targeting peptides for rapid hydrolysis. Both the peptidesα_(S1)-CN(f1-9) and β-CN(f193-209), as well as other related hydrophobicpeptide derivatives, are known to accumulate and have been associatedwith bitter defects in ripened cheeses (Broadbent et al., 2002;Broadbent et al., 1998; Exterkate and Alting, 1995; Kaminogawa et al.,1986; Lee et al., 1996; Lemieux and Simard, 1991). The peptideβ-CN(f193-209) is produced by the activity of chymosin on β-CN.

Interest in the proteolytic system of L. helveticus CNRZ32 is related tothe organism's ability to reduce bitterness and accelerate cheese flavordevelopment when used as an adjunct culture in Gouda cheese production(Bartels et al., 1987a; Bartels et al., 1987b). The ability of Lb.helveticus CNRZ32 to accelerate cheese ripening and reduce bitternesswhen used as an adjunct culture is well documented (Bartels et al.,1987a; Bartels et al., 1987b; Madkor et al., 2000). Lb. helveticusCNRZ32 has been demonstrated to efficiently hydrolyze casein, andcomparison with the peptidolytic activities of Lb. helveticus ATCC 10797and Lactobacillus delbrueckii ssp. bulgaricus ATCC 12278 demonstratedthat Lb. helveticus CNRZ32 had higher general aminopeptidase anddipeptidase activities (Khalid et al., 1991).

The reduction of bitterness in cheese is believed to be the result ofpreferential hydrolysis of low molecular weight hydrophobic peptidesknown to cause bitterness, rather than lack of formation of bitterpeptides from high molecular weight non-bitter casein derived peptides(Broadbent et al., 1998; Gomez et al., 1996; Lee et al., 1996; Lemieuxand Simard, 1991). These bitter peptides contain the amino acid proline,which forms an imino, not amino bond, making these peptides moredifficult to cleave. While numerous enzymes of the proteolytic system ofLb. helveticus have been identified (Christensen et al., 1999), theunderstanding of the specific enzymes responsible for this strain'sability to reduce of bitterness in cheese is incomplete. Thus,identification and characterization of these enzymes are needed.

Moreover, endopeptidases in other contexts have also been explored. Aprolyl endopeptidase was used to reduce the antigencity of a peptideinvolved in celiac sprue, an inflammation of the small intestine (Shanet al. 2002; Vader et al., 2002). Celiac sprue involves gluten peptidesthat survive the digestion process and reach the small intestine becausethey contain proline (see Schuppan et al., 2002). The use of otherprolyl endopeptidases could provide therapeutic benefits for patientswith celiac sprue.

SUMMARY OF THE INVENTION

The present invention is based on the isolation and characterization ofnovel endopeptidases, PepO2 (also referred to as PEPO2) and PepO3 (alsoreferred to as PEPO3), each of which has activity in hydrolyzingproline-containing peptides, such as those that contribute to bittertaste in food, for example, cheese and those involved in celiac sprue.These enzymes have post-proline specificity, which has significantadvantages. The present invention also concerns the isolation andcharacterization of PepF and PepE2, other endopeptidases. Thus, thepresent invention is directed to compositions and methods concerningendopeptidases and their use in hydrolyzing proline-containing peptides.These uses include reducing bitterness in foods whose bitternessinvolves such peptides, and their use in the cheesemaking process.Moreover, these endopeptidases could be employed in other areas in whichcleavage of proline-containing peptides provides a benefit. One sucharea is the treatment or prevention of inflammation, such as in celiacsprue.

Accordingly, compositions of the invention concern nucleic acids,proteinaceous compositions, food additives, vectors, and host cells thatrelate to PepO2, PepO3, PepF, PepN, and/or PepE2. In specificembodiments, it relates to PepO2 and PepO3 from LAB, particularlyLactobacillus, and more particularly, Lactobacillus delbreuckii(including subspecies bulgaricus), Lactobacillus helveticus, andLactobacillus casei. In certain embodiments, compositions furthercomprise PepN, PepF, PepE, PepO and/or PepE2. Any embodiments discussedwith respect to PepO2 may be applied with respect to PepO3, as well asto PepN, PepF, PepE, PepO and/or PepE2, and vice versa. Similarly, anyembodiments discussed with respect to PepO3 may be applied with respectto PepO2, as well as PepN, PepF, PepE, PepO and/or PepE2, and viceversa.

The present invention involves isolated PepO2 polypeptides, which may befull-length, or less or more than full-length. A PepO2 polypeptideincludes a polypeptide with an amino acid sequence that has, or that hasat least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% homology to SEQID NO:2, and in some cases, has the activity of PepO2 from L. helveticusbased on assays as described in the Examples.

A polypeptide has “homology” or is considered “homologous” to a secondpolypeptide if one of the following “homology criteria” is met: 1) atleast 50% of the polypeptide has sequence identity at the same positionswith the second polypeptide; 2) there is some sequence identity at thesame positions with the second polypeptide and at the nonidenticalresidues, at least 50% of them are conservative differences, asdescribed herein, with respect to the second polypeptide; or 3) at least50% of the polypeptide has sequence identity with the secondpolypeptide, but with possible gaps of nonidentical residues betweenidentical residues. If the term “homology” or “homologous” is qualifiedby a number, for example, “80% homology” or “80% homologous,” then thehomology criteria, with respect to 1), 2), and 3), is adjusted from “atleast 50%” to “at least 80%.” Thus it is contemplated that there mayhomology of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or more between two proteinaceous molecules orportions of proteinaceous molecules. It is contemplated that ahomologous polypeptide contains the functional activity of the cognateendopeptidase.

In some embodiments, an isolated PepO2 polypeptide comprises at least 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155,160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225,230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295,300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365,370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435,440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505,510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575,580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, or647 contiguous amino acids of SEQ ID NO:2, and ranges therein. It isspecifically contemplated that an isolated PEPO2 polypeptide comprisesthe amino acid sequence of SEQ ID NO:2.

The present invention also relates to PepO3 polypeptides, which includesa polypeptide with an amino acid sequence that has, or that has at least50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% homology to SEQ ID NO:4,and in some cases, has the activity of PepO3 from Lb. helveticus basedon assays as described in the Examples. In some embodiments, an isolatedPepO3 polypeptide comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115,120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185,190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255,260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325,330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395,400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465,470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535,540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605,610, 615, 620, 625, 630, 635, 640, or 643 contiguous amino acids of SEQID NO:4, and ranges therein. It is specifically contemplated that anisolated PepO3 polypeptide comprises the amino acid sequence of SEQ IDNO:4.

The present invention also relates to PepF. In different embodiments ofthe invention, PepF polypeptides include a polypeptide with an aminoacid sequence that has, or that has at least 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or 100% homology to SEQ ID NO:30, and in some cases, has theactivity of PepF from Lb. helveticus based on assays as described in theExamples. In some embodiments, an isolated PepO3 polypeptide comprisesat least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210,215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280,285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350,355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420,425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490,495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560,565, 570, 575, 580, 585, 590, 595, or 598 contiguous amino acids of SEQID NO:30, and ranges therein. It is specifically contemplated that anisolated PepF polypeptide comprises the amino acid sequence of SEQ IDNO:30.

The present invention also relates to PepE2. In different embodiments ofthe invention, PepE2 polypeptides include a polypeptide with an aminoacid sequence that has, or that has at least 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or 100% homology to SEQ ID NO:32, and in some cases, has theactivity of PepE2 from Lb. helveticus based on assays as described inthe Examples. In some embodiments, an isolated PepE2 polypeptidecomprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130,135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200,205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270,275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340,345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410,415, 420, 425, 430, 435, or 437 contiguous amino acids of SEQ ID NO:32,and ranges therein. It is specifically contemplated that an isolatedPepE2 polypeptide comprises the amino acid sequence of SEQ ID NO:32.

In some embodiments, endopeptidases of the invention have activity withrespect to one or more of SEQ ID NO:5-SEQ ID NO:32, which are peptide orpolypeptide sequences.

The present invention also concerns isolated nucleic acid molecules. Incertain embodiments of the invention, there are isolated PepO2 nucleicacid molecules or polynucleotides comprising a sequence encoding any ofthe polypeptide segments of SEQ ID NO:2 discussed above, or at least orat most of any of the recited segments. Similarly, isolated PepO3nucleic acid molecules or polynucleotides comprise a sequence encodingany of the polypeptide segments of SEQ ID NO:4 discussed above, or atleast or at most of any of the recited segments.

Nucleic acids of the invention also include, or include at least or atmost 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540,550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680,690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820,830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960,970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080,1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200,1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320,1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440,1450, 11500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2395, 2400,2500, 2600, 2700, 2760 contiguous bases or nucleotides of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:31 or SEQ ID NO:33, or any range therein. It isspecifically contemplated that an isolated PepO2 nucleic acid comprisesthe sequence of SEQ ID NO:1. It is also contemplated that an isolatedPepO3 nucleic acid comprises the sequence of SEQ ID NO:3. An isolatedPepF nucleic acid comprises the sequence of SEQ ID NO:31 in someembodiments of the invention, while in others, an isolated PepE2 nucleicacid comprises the sequence of SEQ ID NO:33. It is further contemplatedthat PepO2 and PepO3 nucleic acid molecules have or have at least 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% homology or identity with SEQ ID NO:1 and SEQ ID NO:3,respectively. The term “homology” discussed above can be applied in thecontext of nucleic acids. Alternatively, nucleic acids of the inventioncan also be defined by hybridization characteristics. In someembodiments, nucleic acids of the invention are able to hybridize to SEQID NO:1, SEQ ID NO:3, and/or SEQ ID NO:25-29. Hybridization can occur athigh stringency conditions in some embodiments of the invention.Embodiments regarding Pep-encoding nucleotides such as PepO2 or PepO3also apply to the nucleic acids encoding PepF (SEQ ID NO:31) and PepE2(SEQ ID NO:33) polypeptides disclosed herein.

Furthermore, the present invention concerns compositions with one ormore peptidases. In certain cases, a composition contains 1, 2, 3, 4, 5or more different endopeptidases, including at least one endopeptidasewith prolyl specificity. Any combination of PepO2, PepO3, PepF, PepO,PepE, PepE2, and PepN are contemplated, though compositions are notlimited to only these proteins. These cocktails of endopeptidases can beused to hydrolyze one or more proline-containing peptides. Theirapplication includes, but is not limited to, reduction of bitterness,decontamination measures, and treatment/prevention of celiac sprue. Itis contemplated that such a cocktail contains isolated and activepeptidase(s). “Active” means that the peptidase has the ability tohydrolyze a peptide. In certain cases, it may have a specific level orrange of activities, as defined by endopeptidase units, which isdescribed elsewhere.

The present invention also concerns vectors comprising nucleic acids ofthe invention, as described above and herein. These vectors can havesuch under the control of the promoter and/or have other components ofvectors described herein. It is also contemplated that a single vectormay encode for all or part of SEQ ID NO:2 and/or SEQ ID NO:4. Thus, avector may include sequences encoding for one or more polypeptides.Vectors may include a pepO3 or a pepN promoter in embodiments of theinvention.

Also included as part of the invention are host cells that have one ormore exogenous nucleic acid sequences, particularly those describedabove and herein. In some embodiments, the cell has an exogenous nucleicacid sequence comprising all or part of a PepO2 or PepO3 nucleic acidsequence, such as SEQ ID NO:1 or SEQ ID NO:3. In further embodiments,the cell has an exogenous nucleic acid encoding all or part of a PepO2polypeptide and/or PepO3 polypeptide, such as SEQ ID NO:2 and/or SEQ IDNO:4. It is contemplated that the host cell may be a eukaryotic orprokaryotic cell. In specific embodiments, the host cell is aprokaryotic cell, particularly a bacterial cell. The present inventionmay include the use of any cell used in the cheesemaking process, suchas a starter culture or an adjunct culture. The invention contemplates ahost cell that is a bacterial cell selected from the group consisting ofLactococcus lactis, Streptococcus thermophilus, Lactobacillusdelbreuckii, Lactobacillus helveticus and Lactobacillus casei. Otherhost cells include yeast, fungi and other bacteria. In certainembodiments, a filamentous fungal cell is employed to producepolypeptides of the invention.

In addition to the compositions discussed above, the present inventionconcerns a food additive comprising all or part of a PepO2 and/or PepO3polypeptide, or a nucleic acid encoding such polypeptides. A “foodadditive” refers to a composition that can be added to food or a foodcomponent for consumption by a mammal, such as a human. It iscontemplated that the formulation of the food additive may be as aliquid, power, or solid. It can be used as a food additive during thefood production process or to the ultimate end food product.

The PepO2, PepO3, PepF, and PepE2 polypeptides of the invention as wellas PepN, PepE, and PepO polypeptides that can be used in aspects of theinvention will have activity in many embodiments of the invention. Theiractivity can be expressed in terms of enzyme units. In certainembodiments of the invention, the activity of an endopeptidase of theinvention is about, at least about, or at most about 1, 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 12, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610,620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750,760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890,900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400,2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600,3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800,4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000,6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200,7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400,8500, 8600, 8700, 8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 9600,9700, 9800, 9900, 10000, 11000, 12000, 13000, 14000, 15000, 16000,17000, 18000, 19000, 20000, 21000, 22000, 23000, 24000, 25000, 26000,27000, 28000, 29000, 30000, 31000, 32000, 33000, 34000, 35000, 36000,37000, 38000, 39000, 40000, 41000, 42000, 43000, 44000, 45000, 46000,47000, 48000, 49000, 50000, 60000, 70000, 80000, 90000, 100,000 units ormore, or any range derivable therein. One endopeptidase unit is definedas one nmole of substrate hydrolyzed (β-CN(f193-209)) per hour per mg ofthe protein at 37° C., as described, for example, in Examples 2 and 3.

The invention also concerns methods for producing cheese and forreducing bitterness in food products by cleaving peptides associatedwith a bitter taste. The invention takes advantage of the activity ofPepO2 and/or PepO3 polypeptides in cleaving bitter peptides and thus, itis contemplated that these polypeptides, singly or together or withother endopeptidases, are provided at one or more points during thecheesemaking or other food making process.

In some embodiments, there are methods of producing cheese comprising:a) contacting a bacterial host cell of the invention with milk, wherebyat least one milk product is produced; and, b) producing cheese from themilk product. Thus, in some embodiments, one or more endopeptidases areutilized in the cheesemaking process by providing the endopeptidase withthe starter culture for the cheese or as some other bacterial cell usedin the process. In certain embodiments, the endopeptidase is provided inan adjunct culture, which can be provided after the initial milkproducts are formed. The endopeptidase is provided with the starterculture or other host cell because it exogenously expresses theendopeptidase.

Alternatively, endopeptidases of the invention could be used infoodmaking and cheesemaking processes by adding all or part of theendopeptidase to one or more steps in the process. Therefore, in someembodiments, there are methods for producing cheese involving: a)fermenting a bacterial culture with milk, such that cheese curd isproduced; b) contacting the culture or cheese curd with a PepO2 and/orPepO3 polypeptide; and, c) producing cheese from the product produced byfermentation. One or more endopeptidases could be added to any part ofthe cheesemaking process such as at the beginning, end or during thefollowing: warming milk; pasteurizing or homogenizing milk; coagulatingmilk to produce curds and whey, typically through the exposure of milkto a coagulating agent such as acid or a coagulating enzyme—which isaccomplished by adding a starter culture to milk; adjusting temperatureof milk with starter culture; adding salt; introducing an additive tomilk during curd formation, such as calcium chloride or an antimycoticagent; discarding whey; cutting or breaking curd; cooking curd; drainingor dipping curd; knitting curds; salting curds; pressing curds; or,ripening or curing or aging cheese. It is further contemplated that theymay be added at more than one step during the process or that they maybe added to the ultimate end product. It is also contemplated that otheradditives to reduce bitterness can be introduced into the process. Anystarter culture can be used in the context of the invention and thisincludes, but is not limited to, the following bacteria: Lactococcuslactis, Streptococcus thermophilus, Lactobacillus delbreuckii,Lactobacillus helveticus and Lactobacillus casei.

In further embodiments of the invention, methods include isolating aPepO2 and/or PepO3 polypeptide from a bacterial cell comprising anexogenous nucleic acid sequence encoding a PepO2 and/or PepO3polypeptide(s). The isolated polypeptide can then be added to thecheesemaking or foodmaking process, or be supplied as a food additivecomposition. The polypeptide(s) can be introduced to the starterculture, to the curds, to the whey, or to some other component of theprocess. It is contemplated that at some point subsequent to theintroduction of the endopeptidase polypeptide(s), the polypeptide willbe under conditions that allow it to utilize or exhibit itsendopeptidase activity. In some embodiments, other endopeptidases orother proteins are utilized in these processes. In certain cases, a PepNaminopeptidase is also employed, while in others PepE2, PepO, and/orPepF can be used as well.

Additional embodiments concern a formulation that alleviates or preventsceliac sprue. Compositions of the invention can be used to treat orprevent the inflammation underlying celiac sprue. Thus, methods of theinvention include treating or preventing celiac sprue in a patientcomprising: administering to the patient a pharmaceutically acceptableformulation comprising a PepO2 or PepO3 polypeptide, wherein thepolypeptide has endopeptidase activity. It is contemplated that thepatient may be diagnosed with celiac sprue, have symptoms of celiacsprue, and/or known to be at risk for celiac sprue. In some methods, theformulation comprises both PepO2 and PepO3 polypeptides. In certainembodiments, the formulation is ingested. Furthermore, it may or may notbe ingested at the same time that gluten is ingested. Formulations maybe taken separately from the gluten-containing foodstuff. Alternatively,it may be formulated with the foodstuff or sprinkled on the foodstuff asa powder or liquid. It will be understood that treatment includesreduction or elimination of inflammation, alleviation of symptoms,and/or inhibition of transglutaminase activity or binding.

Other applications include use of one or more endopeptidases of theinvention in a composition that can be used to decontaminate surfaces,such as food preparation surfaces. Proline-containing proteinaceouscompositions can lay on surfaces such that they get transferred tosubjects who may exhibit an allergic reaction to them. Therefore, thepresent invention concerns compositions and methods for decontaminatingan area to reduce or prevent an allergic response to aproline-containing protein. In some embodiments, there is a cleaningsolution or other formulation comprising one or more isolatedendopeptidases selected from the group comprising: PepO2, PepO3, PepF,and PepE2. One or more of the following endopeptidases can also beadded: PepN, Pep 0, and PepE. The formulation may be a solution(concentrated or not) or it may be in a solid form, such as a powder.

Such cleaning solutions or formulations can be used to decontaminate asurface, container, or other area, particularly one involving foodpreparation or used to hold or carry food. The invention also concernsmethods of cleaning an area or surface comprising applying to the areaor surface a composition comprising one or more isolated endopeptidasesselected from the group comprising: PepO2, PepO3, PepF, and PepE2. Incertain embodiments, the solution or formulation comprises a cocktail ofendopeptidases. The solution may be sprayed onto the surface or wipedwith a sponge or other cloth-like material that has the solution on it.

The invention further concerns methods of evaluating endopeptidaseactivity. Such methods of the invention concern an assay forendopeptidase activity under conditions that mimic the cheesemakingprocess, which allows for activity in this particular context to beevaluated. For example, it can be implemented to determine thedebittering efficacy of one or more endopeptidases. The inventioncomprises methods for evaluating endopeptidase activity of anendopeptidase comprising: a) contacting a cheese serum containing apeptide substrate with the endopeptidase; b) measuring hydrolysis of thepeptide substrate. It is contemplated that multiple and differentpeptide substrates may be included or added to the serum. Furthermore, acheddar cheese our gouda serum is particularly contemplated for use withthe method. Such a serum can be routinely prepared by one of skill inthe art, including as described in Example 4 or in Morris et al, whichis specifically incorporated by reference. In some embodiments, a bufferis added to the serum. The serum may be in a concentrated form such as2×, 3×, 4×, 5×, 10×, 15×, 20×, or more. Moreover, the endopeptidase canbe provided as an isolated endopeptidase or in a cell-free extractpreparation. Endopeptidases include, but are not limited to, PepO2,PepO3, PepF, PepO, PepE, PepE2, and PepN, and any combination thereof.

It is specifically contemplated that any embodiments described in theExamples section are included as an embodiment of the invention.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more, unless specifically noted.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Peptide sequences of α_(S1)-CN(f1-9) and β-CN(f193-209) andfragments derived from hydrolysis with CFE's. The residues that areboxed are essential AA for L. helveticus. The shaded residues areessential AA that only occur once in the peptide and therefore must beliberated for growth of Lb. helveticus when the peptide is supplied asthe sole source. The peptides correspond to unique values obtained forRP-HPLC fractions from mass spectrometry data. The strain columnindicates from which CFE reaction(s) a given peptide was identified. Thepeptide profile obtained from hydrolysis of α_(S1)-CN(f1-9) by CFE fromWT is also representative of reactions with ΔpepC, ΔpepO, and ΔpepEstrains. Similarly, the peptide profile obtained from hydrolysis ofβ-CN(f193-209) by CFE from WT is also representative of reactions withthe ΔpepC, ΔpepO, ΔpepE, and ΔpepX strains. Sequences in chart areidentified as follows: (f1-9) (SEQ ID NO:5); f3-9 (SEQ ID NO:6); f4-9(SEQ ID NO:7); f5-9 (SEQ ID NO:8); f1-7 (SEQ ID NO:9); f4-7 (SEQ IDNO:10); f1-5 (SEQ ID NO:11); (f193-209) (SEQ ID NO:12); f194-209 (SEQ IDNO:13); f195-209 (SEQ ID NO:14); f197-209 (SEQ ID NO:15); f199-209 (SEQID NO:16); f201-209 (SEQ ID NO:17); f193-208 (SEQ ID NO:18); f193-206(SEQ ID NO:19); f194-206 (SEQ ID NO:20); f197-206 (SEQ ID NO:21);f199-206 (SEQ ID NO:22); f193-204 (SEQ ID NO:23); f194-204 (SEQ IDNO:24).

FIG. 2A-B. Chromatogram of peptides from hydrolysis of α_(s1)-CN(f1-9)and β-CN(f193-209) by Escherichia coli DH5α (pSUW99). FIG. 2A is achromatogram from the hydrolysis of α_(s1)*CN(f1-9) and FIG. 2B is achromatogram from the hydrolysis of β-CN(f193-209). Hydrolysis reactionswere conducted for 30 min. Major accumulated hydrolysis products areindicated.

FIG. 3. Specificity of PepO2 towards substrates α_(s1)-CN(f1-9) andβ-CN(f193-209). Converging arrows represent the bonds that werehydrolyzed by cell-free extracts of Escherichia coli DH5α (pSUW99),expressing PepO2 from Lactobacillus helveticus, as determined by massspectrometry and calculated molecular mass of peptides derived from bothsubstrates.

FIG. 4A-B. Rate of α_(s1)-CN [f1-9] (FIG. 4A) or β-CN [f193-209] (FIG.4B) hydrolysis under cheese ripening conditions by CFE from E. colitransformants expressing L. helveticus CNRZ32 endopeptidases PepO2(circles), PepO3 (squares), and PepF (triangles). Diamonds show thelevel of background endopeptidase activity in CFE from untransformed E.coli DH5α controls.

FIG. 5. Specificity of Lb. helveticus CNRZ32 endopeptidases towardβ-CN(f193-209) (SEQ ID NO:12) under simulated cheese ripening conditions(pH 5.0-5.2, 4% NaCl, 10° C.).

FIG. 6. Specificity of Lactobacillus helveticus CNRZ32 endopeptidasestoward β-casein (f193-209) (SEQ ID NO:12) and α_(S1)-casein (f1-9) (SEQID NO:5) under cheese ripening conditions (pH 5.0-5.2, 4% NaCl, 10° C.).

FIG. 7. Rate of β-casein (f193-209) (right panel) and α_(s1)-casein(f1-9) (left panel) hydrolysis at pH 5.0-5.2, 4% NaCl at 10° C. insingle peptide system (A, B), defined peptide mix system (C, D), and inCheddar cheese serum (E, F) by cell-free extract from Lactococcus lactisLM0230 derivatives expressing Lactobacillus helveticus CNRZ32endopeptidases PepO2 (circles), PepO3 (squares), and PepE (diamonds).Values are corrected by subtracting the values obtained in the controltreatments. Error bars represent one standard error of the mean (n=3).

FIG. 8. RP-HPLC chromatograms of the products from the hydrolysis ofpeptides in Cheddar cheese serum (CCS) spiked with α_(s1)-casein (f1-9)and β-casein (f193-209) at 10 mg/mL and 1 mg/mL, respectively.Incubations for 12 h with cell free extract of Lactococcus lactis LM0230control (A) and its derivatives expressing Lactobacillus helveticusCNRZ32 PepE (B); PepO3 (C); PepO2 (D) in CCS at 4% NaCl and at pH 5.2and 10° C. Peptides identified in the chromatograms include peak 1,α_(s1)-casein (f1-5); peak 2, α_(s1)-casein (f1-9); peak 3,α_(s1)-casein (f1-13); peak 4, α_(s1)-casein (f4-9); and peak 5,β-casein (f193-209).

FIG. 9. Relative activity of Lactococcus lactis LM0230 derivativesexpressing Lactobacillus helveticus CNRZ32 PepO2, PepO3 and PepE towardspeptides in the defined peptide mix, at pH 5.0-5.2, 4% NaCl, and 10° C.(A) β-casein (f193-209); (B) α_(s1)-casein (f1-9); (C) α_(s1)-casein(f1-6); (D) α_(s1)-casein (f1-13); and (E) α_(s1)-casein (f1-16). Valuesare corrected by subtracting the values obtained in the controltreatments. Activity with α_(s1)-casein (f1-9) was arbitrarily set to100%.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention concerns compositions and methods for producingcheese and/or reducing bitterness in food preparations in whichendopeptidases are involved. More particularly, the invention concernsnucleic acids, proteinaceous compositions, food additives, expressionconstructs, host cells and other compositions described herein, whichmay be used in to reduce bitterness in a number of contexts, includingcheese production.

I. Bacteria Cultures and Endopeptidases

Bitterness in cheese from the presence of bitter peptides is an ongoingand persistent problem. During the cheesemaking process, warm milk isexposed to a starter culture, which typically includes bacteria thatproduce lactic acid. The cultures contain a strain of bacteria capableof producing enzymes that break down proteins in milk, which providesflavor and helps the cheese ripen more quickly. In addition to thestarter culture, cheesemakers sometimes add a bacteria culture toenhance cheese flavor (adjunct culture). Thus, adjunct cultures are alsoused in some cases.

Strains of bacteria used as starter cultures include, but are notlimited to: Lactococcus lactis, Streptococcus thermophilus,Lactobacillus delbreuckii (including subspecies bulgaricus),Lactobacillus helveticus, and Lactobacillus casei. In some cases, thebacteria strain Lactobacillus helveticus is added to the starter cultureto reduce bitterness and enhance flavor. A common adjunct culture isLactobacillus casei.

These bacteria contain endopeptidases, which is an enzyme that cleavesan internal peptide bond (as opposed to a peptide bond on the end of apeptide or polypeptide) and is involved in the cheesemaking process.

In Lactococcus lactis, the best characterized LAB, endopeptidases thathave been identified include PepO, PepO2, PepF1, and PepF2. All of theseenzymes are metalloproteases and PepO, PepF1, and PepF2 are encoded inoperons (Christensen et al., 1999; Kunji et al., 1996). Thephysiological role of these endopeptidases remains unclear; however,PepF appears to be important for protein turnover during nitrogenstarvation (Nardi et al., 1999). To date, one metallo-endopeptidase,designated PepO (Chen and Steele, 1998), and a thiol-dependentendopeptidase, designated PepE (Fenster et al., 1997), have beencharacterized from Lactobacillus helveticus.

L. helveticus peptidases previously investigated include PepC, PepN,PepE, and PepO. They represent three different classes of enzymes. Broadspecificity aminopeptidases (PepC and PepN) remove the N-terminal AAfrom a peptide (X↓Y-Z . . . ), with specificity dependent on the peptidelength and terminal AA residues. X-prolyl dipeptidyl aminopeptidase(PepX) has specificity for removal of proline containing dipeptides(X-Pro↓Y . . . ) from the N-terminus of peptides. Endopeptidases (PepEand PepO) hydrolyze internal peptide bonds ( . . . U-V-W↓X-Y-Z . . . )independent of the N-terminal AA residue, but potentially withspecificity for one or both residues flanking the hydrolyzed peptidebond.

The endopeptidases PepO2 and PepO3 in bacteria, particularly those withcomparable activity and sequence homology to PepO2 and PepO3 from L.helveticus, are specifically contemplated as part of the invention. Thenucleic acid sequence (SEQ ID NO:1) and amino acid sequence (SEQ IDNO:2) for PepO2 in Lactobacillus helveticus CNRZ32 can be found atGenBank Accession number AF321529, which is specifically incorporated byreference. The nucleotide sequence of pepO3, pepF and pepE2 have beendeposited in the GenBank database under accession numbers AY355128,AY365129 and AY365130, respectively, which are hereby incorporated byreference. Each of the accession number discussed in this application isspecifically incorporated by reference.

A. Proteinaceous Compositions

In certain embodiments, the present invention concerns novelcompositions comprising at least one proteinaceous molecule, such as anendopeptidase. In many embodiments, the proteinaceous molecule is all orpart of a PepO2 or PepO3 polypeptide. Moreover, other proteinaceousmolecules may be involved, for example, other enzymes that cleavepeptides involved in bitterness or stabilizing enzymes or otherpolypeptides used in the cheesemaking process. Polypeptides used inmethods of the invention may be produced by recombinant methods, or theymay be naturally produced enzymes, either of which may or may not besubject to subsequent purification or isolation procedures.

As used herein, a “proteinaceous molecule,” “proteinaceous composition,”“proteinaceous compound,” “proteinaceous chain” or “proteinaceousmaterial” generally refers, but is not limited to, a protein of greaterthan about 200 amino acids or the full length endogenous sequencetranslated from a gene; a polypeptide of greater than about 100 aminoacids; and/or a peptide of from about 3 to about 100 amino acids. Allthe “proteinaceous” terms described above may be used interchangeablyherein.

In certain embodiments the size of the at least one proteinaceousmolecule may be at least, at most or may comprise, but is not limitedto, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450,475, 500, 525, 550, 575, 582, 600, 625, 650, 675, 700, 725, 750, 775,800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400,1500, 1750, 2000, 2250, 2500 or greater amino molecule residues, and anyrange derivable therein. It is specifically contemplated that suchlengths of contiguous amino acids from SEQ ID NO:2 or SEQ ID NO:4 arepart of the invention. Moreover, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 or more contiguous amino acids from proteinaceouscompositions of the invention, including such lengths of SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24 (collectively SEQ IDNOs:5-24), are contemplated as part of the invention.

In certain embodiments, an endopeptidase polypeptide contains at leastthe metallo-binding domain of the full-length polypeptide. Other regionsof the enzyme can also be included.

As used herein, an “amino molecule” refers to any amino acid, amino acidderivative or amino acid mimic as would be known to one of ordinaryskill in the art. In certain embodiments, the residues of theproteinaceous molecule are sequential, without any non-amino moleculeinterrupting the sequence of amino molecule residues. In otherembodiments, the sequence may comprise one or more non-amino moleculemoieties. In particular embodiments, the sequence of residues of theproteinaceous molecule may be interrupted by one or more non-aminomolecule moieties.

Proteinaceous compositions may be made by any technique known to thoseof skill in the art, including the expression of proteins, polypeptidesor peptides through standard molecular biological techniques, theisolation of proteinaceous compounds from natural sources, or thechemical synthesis of proteinaceous materials. The nucleotide andprotein, polypeptide and peptide sequences for various genes have beenpreviously disclosed, and may be found at computerized databases knownto those of ordinary skill in the art. One such database is the NationalCenter for Biotechnology Information's Genbank and GenPept databases(found on the World Wide Web at ncbi.nlm.nih.gov/). The coding regionsfor these known genes may be amplified and/or expressed using thetechniques disclosed herein or as would be know to those of ordinaryskill in the art. Alternatively, various commercial preparations ofproteins, polypeptides and peptides are known to those of skill in theart.

1. Functional Aspects

When the present application refers to the function or activity of aendopeptidase, it is meant that the molecule in question has at leastthe ability to hydrolyze nonterminal peptide linkages in an oligopeptideor polypeptide. A synonym for “endopeptidase” is “endoprotease” and theterm “proteinase” includes endopeptidases. Determination of whichmolecules possess this activity and what level of activity there is maybe achieved using assays familiar to those of skill in the art, andinclude those described in the Examples. Christensen et al, 1995a andChristensen et al, 1995b describe methods that can readily be employedto evaluated functional changes, and these references are specificallyincorporated by reference.

2. Peptide Mimetics

Another embodiment for the preparation of polypeptides according to theinvention is the use of peptide mimetics. Mimetics arepeptide-containing molecules that mimic elements of protein secondarystructure. See e.g., Johnson (1993). The underlying rationale behind theuse of peptide mimetics is that the peptide backbone of proteins existschiefly to orient amino acid side chains in such a way as to facilitatemolecular interactions, such as those of antibody and antigen. A peptidemimetic is expected to permit molecular interactions similar to thenatural molecule. These principles may be used, in conjunction with theprinciples outlined above, to engineer second generation moleculeshaving many of the natural properties of an endopeptidase, such as itsspecificity, but with altered and even improved characteristics,including, but not limited to, improved enzyme kinetics, stability, oraddition of other activities or specificities.

3. Fusion Proteins

A specialized kind of insertional variant is the fusion protein, whichis an example of a chimeric polypeptide. This molecule generally has allor a substantial portion of a naturally-occurring polypeptide, linked atthe N- or C-terminus, to all or a portion of a second polypeptide. Forexample, fusions typically employ leader sequences from other species topermit the recombinant expression of a protein in a heterologous host.Another useful fusion includes the addition of a region that facilitatespurification. Inclusion of a cleavage site at or near the fusionjunction will facilitate removal of the extraneous polypeptide afterpurification. Other useful fusions include linking of functionaldomains.

4. Protein Purification

It may be desirable to purify endopeptidases such as PepO2 or PepO3, orvariants thereof. The invention covers addition of one or moreendopeptidases in contemplated methods or as a food additivecomposition. Protein purification techniques are well known to those ofskill in the art. These techniques involve, at one level, the crudefractionation of the cellular milieu to polypeptide and non-polypeptidefractions. Having separated the polypeptide from other proteins, thepolypeptide of interest may be further purified using chromatographicand electrophoretic techniques to achieve partial or completepurification (or purification to homogeneity). Analytical methodsparticularly suited to the preparation of a pure peptide areion-exchange chromatography, exclusion chromatography; polyacrylamidegel electrophoresis; isoelectric focusing. A particularly efficientmethod of purifying peptides is fast protein liquid chromatography oreven HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide. The term “purified protein or peptide” as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the protein or peptide is purified to any degreerelative to its naturally-obtainable state. A purified protein orpeptide therefore also refers to a protein or peptide, free from theenvironment in which it may naturally occur.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulfate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

B. Nucleotides Encoding Endopeptidases

The present invention concerns polynucleotides, isolatable from cells,that are free from total genomic DNA and that are capable of expressingall or part of a protein or polypeptide or that are complementary to allor part of such a polynucleotide. The polynucleotide may encode apeptide or polypeptide containing all or part of a endopeptidase aminoacid sequence or may encode a peptide or polypeptide having all or partof the amino acid sequence of other polypeptides that can be used toreduce bitterness of food products, particularly cheese. Thepolynucleotide may be RNA or DNA.

As used in this application, the term “endopeptidase polynucleotide”refers to a endopeptidase-encoding nucleic acid molecule that has beenisolated free of total genomic nucleic acid. A “PepO2 polynucleotide”refers to a nucleic acid molecule that has been isolated free of totalgenomic DNA and that has the sequence of all or part of a PepO2-encodingnucleic acid sequence. Similarly, a “PepO3 polynucleotide” refers to anucleic acid molecule that has been isolated free of total genomic DNAand that has the sequence of all or part of a PepO3-encoding nucleicacid sequence. A “PepF polynucleotide” refers to a nucleic acid moleculethat has been isolated free of total genomic DNA and that has thesequence of all or part of a PepF-encoding nucleic acid sequence. A“PepE2 polynucleotide” refers to a nucleic acid molecule that has beenisolated free of total genomic DNA and that has the sequence of all orpart of a PepE2-encoding nucleic acid sequence.

A nucleic acid encoding all or part of a native or modified polypeptidemay contain a contiguous nucleic acid sequence encoding all or a portionof such a polypeptide of the following lengths, or be at least or of atmost the following lengths: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380,390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520,530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660,670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800,810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940,950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070,1080, 1090, 1095, 1100, 1500, 2000, 2395, 2500, 3000, 3500, 4000, 4500,5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 10000, or morecontiguous nucleotides, nucleosides, or base pairs of SEQ ID NO:1, SEQID NO:3, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:31, and/or SEQ ID NO:33, as well as any other nucleicacid used as part of the invention.

The present invention concerns nucleic acids capable of hybridizing toSEQ ID NO:1, SEQ ID NO:3, SEQ ID NOs: 25-29, SEQ ID NO:31, and/or SEQ IDNO:33. Accordingly, the nucleotide sequences of the invention may beused for their ability to selectively form duplex molecules withcomplementary stretches of DNAs and/or RNAs or to provide primers foramplification of DNA or RNA from samples. Depending on the applicationenvisioned, one would desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of the probe orprimers for the target sequence.

For applications requiring high selectivity, one will typically desireto employ relatively high stringency conditions to form the hybrids. Forexample, relatively low salt and/or high temperature conditions, such asprovided by about 0.02 M to about 0.10 M NaCl at temperatures of about50° C. to about 70° C. Such high stringency conditions tolerate little,if any, mismatch between the probe or primers and the template or targetstrand and would be particularly suitable for isolating specific genesor for detecting specific mRNA transcripts. It is generally appreciatedthat conditions can be rendered more stringent by the addition ofincreasing amounts of formamide.

For certain applications, for example, site-directed mutagenesis, it isappreciated that lower stringency conditions are preferred. Under theseconditions, hybridization may occur even though the sequences of thehybridizing strands are not perfectly complementary, but are mismatchedat one or more positions. Conditions may be rendered less stringent byincreasing salt concentration and/or decreasing temperature. Forexample, a medium stringency condition could be provided by about 0.1 to0.25 M NaCl at temperatures of about 37° C. to about 55° C., while a lowstringency condition could be provided by about 0.15 M to about 0.9 Msalt, at temperatures ranging from about 20° C. to about 55° C.Hybridization conditions can be readily manipulated depending on thedesired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 1.0 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C.

In particular embodiments, the invention concerns isolated nucleic acidsegments and recombinant vectors incorporating nucleic acid sequencesthat encode a wild-type, altered, or mutant endopeptidases. Thus, anisolated nucleic acid segment or vector containing a nucleic acidsegment may encode, for example, PepO2 or PepO3, which can cleave bitterpeptides. The term “recombinant” may be used in conjunction with apolypeptide or the name of a specific polypeptide, and this generallyrefers to a polypeptide produced from a nucleic acid molecule that hasbeen manipulated in vitro or that is the replicated product of such amolecule.

In other embodiments, the invention concerns isolated nucleic acidsegments and recombinant vectors incorporating nucleic acid sequencesthat encode a polypeptide or peptide that includes within its amino acidsequence a contiguous amino acid sequence in accordance with, oressentially corresponding to the polypeptide.

The nucleic acid segments used in the present invention, regardless ofthe length of the coding sequence itself, may be combined with othernucleic acid sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol.

It is contemplated that the nucleic acid constructs of the presentinvention may encode full-length polypeptide from any source or encode atruncated version of the polypeptide, for example a truncatedendopeptidase polypeptide, such that the transcript of the coding regionrepresents the truncated version. The truncated transcript may then betranslated into a truncated protein. Alternatively, a nucleic acidsequence may encode a full-length polypeptide sequence with additionalheterologous coding sequences, for example to allow for purification ofthe polypeptide, transport, secretion, post-translational modification,or for benefits such as secretion or efficacy. As discussed above, a tagor other heterologous polypeptide may be added to the modifiedpolypeptide-encoding sequence, wherein “heterologous” refers to apolypeptide that is not the same as the modified polypeptide.

1. Vectors

Native and modified polypeptides may be encoded by a nucleic acidmolecule comprised in a vector. The term “vector” is used to refer to acarrier nucleic acid molecule into which a nucleic acid sequence can beinserted for introduction into a cell where it can be replicated. Anucleic acid sequence can be “exogenous,” which means that it is foreignto the cell into which the vector is being introduced or that thesequence is homologous to a sequence in the cell but in a positionwithin the host cell nucleic acid in which the sequence is ordinarilynot found. Vectors include plasmids, cosmids, viruses (bacteriophage,animal viruses, and plant viruses), and artificial chromosomes (e.g.,YACs). One of skill in the art would be well equipped to construct avector through standard recombinant techniques, which are described inSambrook et al. (2001) and Ausubel et al., 1996, both incorporatedherein by reference. In addition to encoding a modified polypeptide, avector may encode non-modified polypeptide sequences such as a tag ortargetting molecule. Useful vectors encoding such fusion proteinsinclude pIN vectors (Inouye et al., 1985), vectors encoding a stretch ofhistidines, and pGEX vectors, for use in generating glutathioneS-transferase (GST) soluble fusion proteins for later purification andseparation or cleavage.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. Expression vectors can contain avariety of “control sequences,” which refer to nucleic acid sequencesnecessary for the transcription and possibly translation of an operablylinked coding sequence in a particular host organism. In addition tocontrol sequences that govern transcription and translation, vectors andexpression vectors may contain nucleic acid sequences that serve otherfunctions as well and are described infra.

There are a number of ways in which expression vectors may be introducedinto cells. In certain embodiments of the invention, the expressionvector comprises a virus or engineered vector derived from a viralgenome. Viral vectors that are specifically contemplated for use withprokaryotic cells are bacteriophage.

a. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind such as RNA polymerase and other transcriptionfactors. The phrases “operatively positioned,” “operatively linked,”“under control,” and “under transcriptional control” mean that apromoter is in a correct functional location and/or orientation inrelation to a nucleic acid sequence to control transcriptionalinitiation and/or expression of that sequence. A promoter may or may notbe used in conjunction with an “enhancer,” which refers to a cis-actingregulatory sequence involved in the transcriptional activation of anucleic acid sequence.

In certain embodiments, an Lb. helveticus promoter can be used toexpress peptidases of the invention. The promoter may be heterologous.In some embodiments, the promoter is a pepO3 promoter or a pepNpromoter.

A promoter may be one naturally associated with a gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment. Such a promoter can be referred to as“endogenous.” Similarly, an enhancer may be one naturally associatedwith a nucleic acid sequence, located either downstream or upstream ofthat sequence. Alternatively, certain advantages will be gained bypositioning the coding nucleic acid segment under the control of arecombinant or heterologous promoter, which refers to a promoter that isnot normally associated with a nucleic acid sequence in its naturalenvironment. A recombinant or heterologous enhancer refers also to anenhancer not normally associated with a nucleic acid sequence in itsnatural environment. Such promoters or enhancers may include promotersor enhancers of other genes, and promoters or enhancers isolated fromany other prokaryotic, viral, or eukaryotic cell, and promoters orenhancers not “naturally occurring,” i.e., containing different elementsof different transcriptional regulatory regions, and/or mutations thatalter expression. In addition to producing nucleic acid sequences ofpromoters and enhancers synthetically, sequences may be produced usingrecombinant cloning and/or nucleic acid amplification technology,including PCR™, in connection with the compositions disclosed herein(see U.S. Pat. Nos. 4,683,202, 5,928,906, each incorporated herein byreference). Furthermore, it is contemplated the control sequences thatdirect transcription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well.

Promoters of specific use are those that may promote transcription in alactic-acid utilizing or producing bacteria, such as those described inU.S. Pat. No. 6,140,078, which is specifically incorporated byreference.

Naturally, it may be important to employ a promoter and/or enhancer thateffectively directs the expression of the DNA segment in the cell type,organelle, and organism chosen for expression. Those of skill in the artof molecular biology generally know the use of promoters, enhancers, andcell type combinations for protein expression, for example, see Sambrooket al. (2001), incorporated herein by reference. The promoters employedmay be constitutive, inducible, and/or useful under the appropriateconditions to direct high level expression of the introduced DNAsegment, such as is advantageous in the large-scale production ofrecombinant proteins and/or peptides. The promoter may be heterologousor endogenous.

It is specifically contemplated that nucleic acid sequences of interestmay be expressed in fungus and in lactococci bacteria, such as Lc.Lactis, a commonly used cheese starter. Thus, promoters that can directexpression in these organisms are specifically contemplated. Suchpromoters include the pepO3 promoter discussed herein, which can be usedin lactococci.

b. Initiation Signals

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

c. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector. (See Carbonelli et al., 1999, Levenson et al., 1998,and Cocea, 1997, incorporated herein by reference.) “Restriction enzymedigestion” refers to catalytic cleavage of a nucleic acid molecule withan enzyme that functions only at specific locations in a nucleic acidmolecule. Many of these restriction enzymes are commercially available.Use of such enzymes is widely understood by those of skill in the art.Frequently, a vector is linearized or fragmented using a restrictionenzyme that cuts within the MCS to enable exogenous sequences to beligated to the vector. “Ligation” refers to the process of formingphosphodiester bonds between two nucleic acid fragments, which may ormay not be contiguous with each other. Techniques involving restrictionenzymes and ligation reactions are well known to those of skill in theart of recombinant technology.

d. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

e. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

f. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, ampicillin, GPT, zeocinand histidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as lacZ, whose basis iscolorimetric analysis, are also contemplated. The marker used is notbelieved to be important, so long as it is capable of being expressedsimultaneously with the nucleic acid encoding a gene product. Furtherexamples of selectable and screenable markers are well known to one ofskill in the art.

2. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organisms that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid, such as a modified protein-encoding sequence, istransferred or introduced into the host cell. A transformed cellincludes the primary subject cell and its progeny.

Host cells may be derived from prokaryotes or eukaryotes, includingbacteria cells, insect cells, and mammalian cells, depending uponwhether the desired result is replication of the vector or expression ofpart or all of the vector-encoded nucleic acid sequences. Numerous celllines and cultures are available for use as a host cell, and they can beobtained through the American Type Culture Collection (ATCC), which isan organization that serves as an archive for living cultures andgenetic materials (www.atcc.org). An appropriate host can be determinedby one of skill in the art based on the vector backbone and the desiredresult. A plasmid or cosmid, for example, can be introduced into aprokaryote host cell for replication of many vectors. Bacterial cellsused as host cells for vector replication and/or expression includeDH5α, JM109, and KC8, as well as a number of commercially availablebacterial hosts such as SURE® Competent Cells and SOLOPACK™ Gold Cells(STRATAGENE®, La Jolla, Calif.). Bacteria can also be used forlarge-scale production. In some embodiments, a prokaryotic cells isBacillus subtilis or Streptococcus lividans.

Alternatively, bacterial cells such as E. coli LE392 could be used ashost cells for phage viruses. In other embodiments, bacteria used as astarter culture or as an additive to a starter culture serve as hostcells for vectors or plasmids encoding all or part of an endopeptidase,such as PEPO2 and/or PEPO3.

Cells for the commercial production of polypeptides are alsocontemplated. In addition to mammalian cells, yeast, fungi and bacterialcells have been used. See Benyx, 2004. Yeast include but are not limitedto methylotrophic yeast such as Pichia pastoris and Pichia methanolica.Fungi can be used, particularly filamentous fungi. In certainembodiments, a fungus such as Chrysosporium lucknowense (C1), Apergillusniger, and Trichoderma reesei can be employed as a host cell. Alsocontemplated are Penicillium roqueforti, Penicillium candidum,Geotrichum candidum, Torula kefir and Saccharomyces kefir. Theseapplications are well known to those of skill in the art and aredescribed in the literature, for example, in the Handbook of FungalBiotechnology (Arora et al., 2003) and Punt et al., 2002, which arespecifically incorporated by reference. Moreover, thermofilic fungi canbe employed: Zygomycetes; Absidia corymbifera; Mortierella turficola; M.wolfi; Mucor miehei; M. pusillus; Rhizomucor sp.; Rhizopus arrhizus; R.cohnii; R. microsporus; Ascomycetes; Allescheria terrestris;Byssochlamys verrucosa; Chaetomium britannicum; C. thermophile; C.thermophile var. coprophile; C. thermophile var. dissitum; C.virginicum; Emericella nidulans; Hansenula polymorpha; Myriococcumalbomyces; Sphaerospora saccata; Talaromyces byssochlamydoides; T.emersonii; T. leycettanus; T. thermophilus; Thermoascus aurantiacus; T.crustaceus; Thielavia australiensis; T. sepedonium; T. thermophila;Basidiomycetes; Coprinus delicatulus; Mycelia Sterila;Burgoa-Papulaspora; Papulaspora thermophila; Deuteromycetes; Acremoniumalbamensis; Acrophialophora fusispora; Aspergillus candidus; A.fumigatus; Botrytis sp. (=Sphaerospora saccata); Calcarisporiumthermophile; Cephalosporium sp. (=Allescheria terrestris);Cephalosporium sp. (=Thielavia australiensis); Geotrichum sp. A;Humicola grisea var. thermoidea; H. insolens; H. lanuginosa; H.stellata; Malbranchea pulchella var. sulfurea; Nodulisporiumcylindroconium (Tritirachium sp. A); Paecilomyces crustaceus(=Thermoascus); P. puntonii; P. variotii; Paecilomyces sp.(=Byssochlamys verrucosa); Paecilomyces sp. (=Talaromycesbyssochlamydoides); Penicillium duponti (=Talaromyces thermophilus); P.emersonii (=Talaromyces); P. leycettanum (=Talaromyces); P. piceum; P.argillaceum; Ptychogaster sp. (Sporotrichum pulverulentum);Scolecobasidium sp. A (=Diplorhinotrichum galloparum); Sporotrichumthermophile (=Thielavia); S. pulverulentum; Stilbella thermophila;Thermomyces ibadanesis; Torula thermophila; Torulopsis candida;Tritirachium sp. A (=Nodulisporium cylindroconium). The choice of aspecies, and within a species the choice of a strain or variety, whichwill accomplish the desired result and will produce endopeptidases atacceptable yields and having adequate thermal stability and activity, isa process of systematic testing and assay procedures. Once asatisfactory species and a satisfactory strain or variety of thatspecies have been provided, it will provide a suitable continuing sourceof the organism for the production of lactase in yields and of qualitiesdesired.

Other examples for expression of endopeptidases of the invention and theuse of starter cultures can be found in U.S. Pat. Nos. 6,548,089,6,335,040, 6,127,142, and 5,888,966, which are hereby incorporated byreference for this information, as well as embodiments regardingformulations and other cheesemaking processes.

3. Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with the present invention to producenucleic acid sequences, or their cognate polypeptides, proteins andpeptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986, 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the nameMAXBAC®2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEMFROM CLONTECH®.

In addition to the disclosed expression systems of the invention, otherexamples of expression systems include STRATAGENE®'S COMPLETE CONTROL™Inducible Mammalian Expression System, which involves a syntheticecdysone-inducible receptor, or its pET Expression System, an E. coliexpression system.

4. Methods of Nucleic Acid Transfer

Suitable methods for nucleic acid delivery to effect expression ofcompositions of the present invention are believed to include virtuallyany method by which a nucleic acid (e.g., DNA, including viral andnonviral vectors) can be introduced into an organelle, a cell, a tissueor an organism, as described herein or as would be known to one ofordinary skill in the art. Such methods include, but are not limited to,direct delivery of DNA such as by injection (U.S. Pat. Nos. 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610,5,589,466 and 5,580,859, each incorporated herein by reference),including microinjection (Harland and Weintraub, 1985; U.S. Pat. No.5,789,215, incorporated herein by reference); by electroporation (U.S.Pat. No. 5,384,253, incorporated herein by reference); by calciumphosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama,1987; Rippe et al., 1990); by using DEAE-dextran followed bypolyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimeret al., 1987); by liposome mediated transfection (Nicolau and Sene,1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980;Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment(PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat. Nos.5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880, andeach incorporated herein by reference); by agitation with siliconcarbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and5,464,765, each incorporated herein by reference); bydesiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985).Through the application of techniques such as these, organelle(s),cell(s), tissue(s) or organism(s) may be stably or transientlytransformed.

II. Methods of Producing Cheese

Methods of the invention involve using endopeptidases to reducebitterness. In specific embodiments, methods concern usingendopeptidases in the production of cheese, which can become bitterduring the aging process.

The production of cheese involves a number of steps, which may include,but are not limited to, one or more of the following steps (whose ordermay be altered): warming milk; pasteurizing or homogenizing milk;coagulating milk to produce curds and whey, typically through theexposure of milk to a coagulating agent such as acid or a coagulatingenzyme—which is accomplished by adding a starter culture to milk;adjusting temperature of milk with starter culture; adding salt;introducing an additive to milk during curd formation, such as calciumchloride or an antimycotic agent; discarding whey; cutting or breakingcurd; cooking curd; draining or dipping curd; knitting curds; saltingcurds; pressing curds; and, ripening or curing or aging cheese at aparticular temperature and humidity level for a particular amount oftime. Moreover, in particular embodiments, methods of the inventioninvolve traditional or well known cheese-making steps but also includesteps and compositions involving endopeptidases of the invention. Stepsconcerning endopeptidases may involve introduction of the enzymesbatchwise, e.g., in a tank with stirring, or the introduction may becontinuous, e.g., a series of stirred tank reactors.

Methods of cheesemaking are well known to those of skill in the art.Such methods include those described in the following patents, which arehereby incorporated by reference: U.S. Pat. Nos. 6,572,901; 6,558,716;6,551,635; 6,548,089; 6,485,762; 6,475,538; 6,468,570; 6,465,033;6,458,394; 6,455,092; 6,443,379; 6,416,797; 6,413,568; 6,410,076;6,401,604; 6,399,121; 6,335,040; 6,297,042; 6,270,823; 6,258,390;6,242,036; 6,183,804; 6,140,078; 6,139,889; 6,120,809; 6,103,277;6,026,740; 5,988052; 5.948,459; 5,853,786; 5,688,542; 5,643,621;5,635,228; 5,554,398; 5,547,691; 5,505,979; 5,462,755; 5,429,829;5,395,631; 5,356,639; 5,106,631.

Production of the following cheeses is specifically contemplated, thoughthe invention is not limited to their production: Abbaye de Belloc,Abbaye du Mont des Cats, Abertam, Abondance, Ackawi, Acorn, Adelost,Affidelice au Chablis, Afuega'l Pitu, Airag, Airedale, Aisy Cendre,Allgauer Emmentaler, Alverca, Ambert, American Cheese, Ami duChambertin, Anejo Enchilado, Anneau du Vic-Bilh, Anthoriro, Appenzell,Aragon, Ardi Gasna, Ardrahan, Armenian String, Aromes au Gene de Marc,Asadero, Asiago, Aubisque Pyrenees, Autun, Avaxtskyr, Baby Swiss,Babybel, Baguette Laonnaise, Bakers, Baladi, Balaton, Bandal, Banon,Barry's Bay Cheddar, Basing, Basket Cheese, Bath Cheese, BavarianBergkase, Baylough, Beaufort, Beauvoorde, Beenleigh Blue, Beer Cheese,Bel Paese, Bergader, Bergere Bleue, Berkswell, Beyaz Peynir, Bierkase,Bishop Kennedy, Blarney, Bleu d'Auvergne, Bleu de Gex, Bleu deLaqueuille, Bleu de Septmoncel, Bleu Des Causses, Blue, Blue Castello,Blue Rathgore, Blue Vein (Australian), Blue Vein Cheeses, Bocconcini,Bocconcini (Australian), Boeren Leidenkaas, Bonchester, Bosworth,Bougon, Boule Du Roves, Boulette d'Avesnes, Boursault, Boursin,Bouyssou, Bra, Braudostur, Breakfast Cheese, Brebis du Lavort, Brebis duLochois, Brebis du Puyfaucon, Bresse Bleu, Brick, Brie, Brie de Meaux,Brie de Melun, Brillat-Savarin, Brin, Brin d'Amour, Brin d'Amour, Brinza(Burduf Brinza), Briquette de Brebis, Briquette du Forez, Broccio,Broccio Demi-Affine, Brousse du Rove, Bruder Basil, Brusselae Kaas(Fromage de Bruxelles), Bryndza, Buchette d'Anjou, Buffalo, Burgos,Butte, Butterkase, Button (Innes), Buxton Blue, Cabecou, Caboc,Cabrales, Cachaille, Caciocavallo, Caciotta, Caerphilly, Cairnsmore,Calenzana, Cambazola, Camembert de Normandie, Canadian Cheddar,Canestrato, Cantal, Caprice des Dieux, Capricorn, Goat, Capriole Banon,Carre de l'Est, Casciotta di Urbino, Cashel Blue, Castellano,Castelleno, Castelmagno, Castelo Branco, Castigliano, Cathelain, CelticPromise, Cendre d'Olivet, Cerney, Chabichou, Chabichou du Poitou, Chabisde Gatine, Chaource, Charolais, Chaumes, Cheddar, Cheshire, Chevres,Chevrotin des Aravis, Chontaleno, Civray, Coeur de Camembert auCalvados, Coeur de Chevre, Colby, Cold Pack, Comte, Coolea, Cooleney,Coquetdale, Corleggy, Cornish Pepper, Cotherstone, Cotija, CottageCheese, Cottage Cheese (Australian), Cougar Gold, Coulommiers,Coverdale, Crayeux de Roncq, Cream Cheese, Cream Havarti, Crema Agria,Crema Mexicana, Creme Fraiche, Crescenza, Croghan, Crottin de Chavignol,Crottin du Chavignol, Crowdie, Crowley, Cuajada, Curd, Cure Nantais,Curworthy, Cwmtawe Pecorino, Cypress Grove Chevre, Danablu (DanishBlue), Danbo, Danish Fontina, Daralagjazsky, Dauphin, Delice desFiouves, Denhany Dorset Drum, Derby, Dessertnyj Belyj, Devon Blue, DevonGarland, Dolcelatte, Doolin, Doppelrhamstufel, Dorset Blue Vinney,Double Gloucester, Double Worcester, Dreux a la Feuille, Dry Jack,Duddleswell, Dunbarra, Dunlop, Dunsyre Blue, Duroblando, Durrus, DutchMimolette (Commissiekaas), Edam, Edelpilz, Emental Grand Cru, Emlett,Emmental, Epoisses de Bourgogne, Esbareich, Esrom, Etorki, EvansdaleFarmhouse Brie, Evora De L'Alentejo, Exmoor Blue, Explorateur, Feta,Feta (Australian), Figue, Filetta, Fin-de-Siecle, Finlandia Swiss, Finn,Fiore Sardo, Fleur du Maquis, Flor de Guia, Flower Marie, Folded, Foldedcheese with mint, Fondant de Brebis, Fontainebleau, Fontal, Fontina ValD'Aosta, Fougerus, Four Herb Gouda, Fourme d'Ambert, Fourme de HauteLoire, Fourme de Montbrison, Fresh Jack, Fresh Mozzarella, FreshRicotta, Fresh Truffles, Fribourgeois, Friesekaas, Friesian, Friesla,Frinault, Fromage a Raclette, Fromage Corse, Fromage de Montagne deSavoie, Fromage Frais, Fruit Cream Cheese, Frying Cheese, Fynbo,Gabriel, Galette du Paludier, Galette Lyonnaise, Galloway Goat's MilkGems, Gammelost, Gaperon a l'Ail, Garrotxa, Gastanberra, Geitost,Gippsland Blue, Gjetost, Gloucester, Golden Cross, Gorgonzola,Gornyaltajski, Gospel Green, Gouda, Goutu, Gowrie, Grabetto, Graddost,Grafton Village Cheddar, Grana, Grana Padano, Grand Vatel, Gratarond'Areches, Gratte-Paille, Graviera, Greuilh, Greve, Gris de Lille,Gruyere, Gubbeen, Guerbigny, Halloumi, Halloumy (Australian),Haloumi-Style Cheese, Harbourne Blue, Havarti, Heidi Gruyere, HerefordHop, Herrgardsost, Herriot Farmhouse, Herve, Hipi Iti, Hubbardston BlueCow, Hushallsost, Iberico, Idaho Goatster, Idiazabal, Il Boschetto alTartufo, Ile d'Yeu, Isle of Mull, Jarlsberg, Jermi Tortes, JibnehArabieh, Jindi Brie, Jubilee Blue, Juustoleipa, Kadchgall, Kaseri,Kashta, Kefalotyri, Kenafa, Kernhem, Kervella Affine, Kikorangi, KingIsland Cape Wickham Brie, King River Gold, Klosterkaese, Knockalara,Kugelkase, L'Aveyronnais, L'Ecir de l'Aubrac, La Taupiniere, La VacheQui Rit, Laguiole, Lairobell, Lajta, Lanark Blue, Lancashire, Langres,Lappi, Laruns, Lavistown, Le Brin, Le Fium Orbo, Le Lacandou, Le Roule,Leafield, Lebbene, Leerdammer, Leicester, Leyden, Limburger,Lincolnshire Poacher, Lingot Saint Bousquet d'Orb, Liptauer, LittleRydings, Livarot, Llanboidy, Llanglofan Farmhouse, Loch ArthurFarmhouse, Loddiswell Avondale, Longhorn, Lou Palou, Lou Pevre,Lyonnais, Maasdam, Macconais, Mahoe Aged Gouda, Mahon, Malvern,Mamirolle, Manchego, Manouri, Manur, Marble Cheddar, Marbled Cheeses,Maredsous, Margotin, Maribo, Maroilles, Mascares, Mascarpone, Mascarpone(Australian), Mascarpone Torta, Matocq, Maytag Blue, Meira, MenallackFarmhouse, Menonita, Meredith Blue, Mesost, Metton (Cancoillotte), MeyerVintage Gouda, Mihalic Peynir, Milleens, Mimolette, Mine-Gabhar, MiniBaby Bells, Mixte, Molbo, Monastery Cheeses, Mondseer, Mont D'orLyonnais, Montasio, Monterey Jack, Monterey Jack Dry, Morbier, MorbierCru de Montagne, Mothais a la Feuille, Mozzarella, Mozzarella(Australian), Mozzarella di Bufala, Mozzarella Fresh, in water,Mozzarella Rolls, Munster, Murol, Mycella, Myzithra, Naboulsi, Nantais,Neufchatel, Neufchatel (Australian), Niolo, Nokkelost, Northumberland,Oaxaca, Olde York, Olivet au Foin, Olivet Bleu, Olivet Cendre, OrkneyExtra Mature Cheddar, Orla, Oschtjepka, Ossau Fermier, Ossau-Iraty,Oszczypek, Oxford Blue, P'tit Berrichon, Palet de Babligny, Paneer,Panela, Pannerone, Pant ys Gawn, Parmesan (Parmigiano), ParmigianoReggiano, Pas de l'Escalette, Passendale, Pasteurized Processed, Pate deFromage, Patefine Fort, Pave d'Affinois, Pave D'Auge, Pave de Chirac,Pave du Berry, Pecorino, Pecorino in Walnut Leaves, Pecorino Romano,Peekskill Pyramid, Pelardon des Cevennes, Pelardon des Corbieres,Penamellera, Penbryn, Pencarreg, Perail de Brebis, Petit Morin, PetitPardou, Petit-Suisse, Picodon de Chevre, Picos de Europa, Piora,Pithtviers au Foin, Plateau de Herve, Plymouth Cheese, Podhalanski,Poivre d'Ane, Polkolbin, Pont l'Eveque, Port Nicholson, Port-Salut,Postel, Pouligny-Saint-Pierre, Pourly, Prastost, Pressato, Prince-Jean,Processed Cheddar, Provolone, Provolone (Australian), Pyengana Cheddar,Pyramide, Quark, Quark (Australian), Quartirolo Lombardo, Quatre-Vents,Quercy Petit, Queso Blanco, Queso Blanco con Frutas—Pina y Mango, Quesode Murcia, Queso del Montsec, Queso del Tietar, Queso Fresco, QuesoFresco (Adobera), Queso Iberico, Queso Jalapeno, Queso Majorero, QuesoMedia Luna, Queso Para Frier, Queso Quesadilla, Rabacal, Raclette,Ragusano, Raschera, Reblochon, Red Leicester, Regal de la Dombes,Reggianito, Remedou, Requeson, Richelieu, Ricotta, Ricotta (Australian),Ricotta Salata, Ridder, Rigotte, Rocamadour, Rollot, Romano, Romans PartDieu, Roncal, Roquefort, Roule, Rouleau De Beaulieu, Royalp Tilsit,Rubens, Rustinu, Saaland Pfarr, Saanenkaese, Saga, Sage Derby, SainteMaure, Saint-Marcellin, Saint-Nectaire, Saint-Paulin, Salers, Samso, SanSimon, Sancerre, Sap Sago, Sardo, Sardo Egyptian, Sbrinz, Scamorza,Schabzieger, Schloss, Selles sur Cher, Selva, Serat, Seriously StrongCheddar, Serra da Estrela, Sharpam, Shelburne Cheddar, Shropshire Blue,Siraz, Sirene, Smoked Gouda, Somerset Brie, Sonoma Jack, Sottocenare alTartufo, Soumaintrain, Sourire Lozerien, Spenwood, SraffordshireOrganic, St. Agur Blue Cheese, Stilton, Stinking Bishop, String, SussexSlipcote, Sveciaost, Swaledale, Sweet Style Swiss, Swiss Syrian(Armenian String), Tala, Taleggio, Tamie, Tasmania Highland Chevre Log,Taupiniere, Teifi, Telemea, Testouri, Tete de Moine, Tetilla, Texas GoatCheese, Tibet, Tillamook Cheddar, Tilsit, Timboon Brie, Toma, TommeBrulee, Tomme d'Abondance, Tomme de Chevre, Tomme de Romans, Tomme deSavoie, Tomme des Chouans, Tommes, Torta del Casar, Toscanello, Toureede L'Aubier, Tourmalet, Trappe (Veritable), Trois Comes De Vendee,Tronchon, Trou du Cru, Truffe, Tupi, Turunmaa, Tymsboro, Tyn Grug,Tyning, Ubriaco, Ulloa, Vacherin-Fribourgeois, Valencay,Vasterbottenost, Venaco, Vendomois, Vieux Corse, Vignotte, Vulscombe,Waimata Farmhouse Blue, Washed Rind Cheese (Australian), Waterloo,Weichkaese, Wellington, Wensleydale, White Stilton, WhitestoneFarmhouse, Wigmore, Woodside Cabecou, Xanadu, Xynotyro, Yarg Cornish,Yarra Valley Pyramid, Yorkshire Blue, Zamorano, Zanetti Grana Padano,Zanetti Parmigiano Reggiano.

III. Formulations for Food Additives

Formulations for food additives are well known to those of skill in theart. The additives of the invention may be formulated as a liquid orsolid pellet, powder, spray, or tablet capsule.

The use of a proteinaceous composition in a liquid composition is wellknow. Compositions can include glycerol, including 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% or more glycerol(v/v). Compositions may also include stabilizers or carrier proteins,such as albumin, or preservatives.

Various other additives which are conventionally added to enzyme foodsupplement compositions, such as preservatives and the like, may beutilized. U.S. Pat. Nos. 5,387,422, 6,020,324 and 6,299,896 discuss foodadditive formulations and are specifically incorporated by reference.

The compositions disclosed herein may be delivered via oraladministration to a person, and as such, these compositions may beformulated with an inert diluent or with an assimilable edible carrier,or they may be enclosed in hard- or soft-shell gelatin capsule, or theymay be compressed into tablets, or they may be incorporated directlywith the food of the diet.

The active compounds may even be incorporated with excipients and usedin the form of ingestible tablets, buccal tables, troches, capsules,elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al.,1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and5,792,451, each specifically incorporated herein by reference in itsentirety). The tablets, troches, pills, capsules and the like may alsocontain the following: a binder, as gum tragacanth, acacia, cornstarch,or gelatin; excipients, such as dicalcium phosphate; a disintegratingagent, such as corn starch, potato starch, alginic acid and the like; alubricant, such as magnesium stearate; and a sweetening agent, such assucrose, lactose or saccharin may be added or a flavoring agent, such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup of elixir may contain the active compoundssucrose as a sweetening agent methyl and propylparabens aspreservatives, a dye and flavoring, such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparation and formulations.

Typically, these formulations may contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

The phrase “pharmacologically acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

III. Formulations for Cleaning Solutions and Formulations

Formulations for cleaning compositions are well known to those of skillin the art. Such formulations may be solid or liquid, and can morespecifically be a solid, pellet, powder, or spray. Examples of suchformulations and other components of such compositions can be found, forexample, in U.S. Pat. Nos. 6,686,324, 6,475,290, 6,066,610, 5,776,351,5,593,598, 5,469,880, 5,234,268, 5,020,917, 4,739,906, 4,063,893,4,017,410, and 4,001,133, which are specifically incorporated byreference.

IV. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Hydrolysis of Casein Derived Peptides by L. HelveticusPeptidase Deletion Mutants Indicates Presence of Previously UndetectedEndopeptidase

A. Growth in AA Defined Media and Defined Media with Peptide Supplements

To examine effects of peptidase mutations on growth in media requiringhydrolysis of exogenous peptides, growth of L. helveticus and thepeptidase deletion mutants (Table 1) were determined in defined mediaprepared with either α_(s1)-CN(f1-9) or β-CN(f193-209) as the solesource of several essential amino acids (AA). Growth in media notrequiring hydrolysis of exogenous peptides to obtain AA was determinedin defined media (Christensen, 2000). The defined media is not minimaland is comprised of all free AA as the sole nitrogen source (includingthe non-essential Ala, Asn, Gln, Gly, Cys and Ser).

The defined media supplemented with α_(s1)-CN(f1-9) was prepared withall the components of complete defined media except the essential AAcontained within this peptide (Arg, His, Ile, Lys, and Pro) (Table 1).Likewise, the defined media supplemented with β-CN(f193-209) wasprepared with all the components of complete defined media except theessential AA contained within this peptide (Arg, Ile, Leu, Phe, Pro,Tyr, and Val). Inoculations were prepared from cultures propagated inMRS at 42° C. to late exponential phase. Cells were washed andresuspended in 0.85% NaCl to the original volume, defined media wasinoculated at about 10⁶ cell/ml, and cultures were incubated at 42° C.for 18 hours.

There were no discernable differences in growth for any of the strainsin AA defined media or peptide supplemented media (all strains OD₆₀₀about 2.4-2.8, final pH˜3.6). The lack of growth deficiencies of singlepeptidase deletion mutant strains (ΔpepC, ΔpepE, ΔpepN, ΔpepO, or ΔpepX)in defined peptide media indicates that the target peptidases are notnormally involved in the hydrolysis of these peptides and/or theessential AA is liberated efficiently via an alternative pathway ofhydrolysis in the absence of a given peptidase.

Complete hydrolysis of the peptide substrates is not necessarilyrequired in order for L. helveticus to grow in the described definedpeptide media. Some of the AA essential to L. helveticus occur only oncein α_(s1)-CN(f1-9) (Arg & Ile) and β-CN(f193-209) (Tyr, Leu, Arg, & Phe)and therefore must be liberated for growth in the defined media (Table2). However, there are two residues of each of the remaining essentialAAs per peptide in α_(s1)-CN(f1-9). In β-CN(f193-209), there aremultiple residues per peptide for Pro (four), Val (three), and Ile(two).

TABLE 1 Bacterial Strains Strain Relevant feature(s) Source or referenceCNRZ32 Wild type; auxotrophic for all Laboratory strain AA except Ala,Asn, Cys, Gln, Gly, and Ser. JLS241 ΔpepC derivative of CNRZ32(Christensen, 2000) JLS242 ΔpepN derivative of CNRZ32 (Christensen,2000) JLS243 ΔpepX derivative of CNRZ32 (Christensen, 2000) JLS233 ΔpepEderivative of CNRZ32 (Fenster and Steele, 2000) JLS232 ΔpepO derivativeof CNRZ32 (Chen and Steele, 1998)B. Hydrolysis of Peptides by De-energized Whole Cells

With respect to the growth analysis of the CFE derived peptides, thepossibility that the substrates undergo initial hydrolysis by a cellenvelope proteinase (CEP) prior to transport of the resulting peptideswas also investigated. De-energized whole cell suspensions were preparedand incubated with the peptides under the same conditions described forCFE reactions and the reaction supernatants were analyzed by RP-HPLC(see methods below). No evidence of extracellular hydrolysis was foundfrom extended time reactions (120 min) of the peptides with whole cells.Although this result was not expected, it suggests that extracellularhydrolysis prior to transport is not necessary for these peptides. Thereis a precedent for other multiply AA auxotrophic LAB to transport evenlarger peptides via the oligopeptide transport system (Opp) thanreported for homologues in bacteria with fewer AA auxotrophies (Payne,1968; Perego et al., 1991). Kinetic analysis of Opp from Lactococcusindicates that peptides from four to at least eighteen residues can betransported with little specificity for particular AA side chains(Detmers et al., 1998). Therefore, it is a formal possibility the L.helveticus is capable of transporting α_(s1)-CN(f1-9) and β-CN(f193-209)without prior hydrolysis.

C. Hydrolysis of α_(s1)-CN(f1-9) and β-CN(f193-209) with CFE of L.helveticus and Peptidase Mutants

The peptides α_(s1)-CN(f1-9) and β-CN(f193-209) were synthesized andpurified at the Utah State University Biotechnology Center. Cell freeextracts (CFE) were prepared as described previously (Christensen,2000). The protein concentration of the CFE and bovine serum albuminstandards was determined using a bicinchoninic acid assay kit (Sigma,St. Louis, Mo.) (Smith et al., 1985).

Peptide hydrolysis reactions with L. helveticus CFE were performed in 50μl total volumes. Each sample contained 45 μl of CFE or appropriatedilution to 0.95-1.05 mg/ml protein. The reactions commenced with theaddition of the peptide substrate and were incubated at 37° C. Initialsubstrate concentrations in the reaction samples were 5.0 μg/l forα_(s1)-CN(f1-9), β-CN(f193-209), and β-CN(f193-209-n-butyl amide).Incubation times of 40 min were determined to result in hydrolysisproducts of relatively even distribution and approximately 25-30% (bypeak area) of the original peptide substrates remained. Reactions werestopped by addition of 200 μl of 40% (v/v) MeCN. Sample stability wasdetermined to be >12 hr at room temperature.

Hydrolysis samples were separated and collected from RP-HPLC asdescribed in Chen et al. (Chen et al., 2002). The mass of RP-HPLCseparated peptide fractions were determined using a Perkin Elmer API 365triple quadrupole electrospray ionization mass spectrometer (ESIMS) or aBruker Reflex II for matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF-MS). Peptides that could notbe positively identified by mass alone were analyzed by ESIMS/MS tomeasure fragments of the parent peptide. ESIMS was performed at theBiotechnology Center and MALDI-TOF-MS was performed and the ChemistryInstrument Center, both at the University of Wisconsin—Madison.

The reactions of α_(s1)-CN(f1-9) or β-CN(f193-209) with CFE of L.helveticus wild type and the peptidase deletion mutants wereinvestigated to assess hydrolytic differences due to the absence of agiven peptidase. The rate of depletion of α_(S1)-CN(f1-9) and theaccumulation of the primary hydrolysis product, α_(S1)-CN(f1-7), wasindistinguishable for all six strains. However, the remaining peaksaccumulated from for hydrolysis by ΔpepN or ΔpepX strains were differentfrom each other and different from wild type (as well as ΔpepC, ΔpepO,and ΔpepE strains; see FIG. 1).

The chromatograms for reactions of β-CN(f193-209) with CFE from wildtype, ΔpepC, ΔpepO, ΔpepE, and ΔpepX strains were indistinguishable interms of the rate of hydrolysis and the accumulation of the alldetectable peptides (represented by WT in FIG. 1). However, thechromatogram peak profiles from hydrolysis with the ΔpepN strain weredistinctly different. The most notable differences in the reaction withthe ΔpepN strain were the decreased rate of hydrolysis of the initialsubstrate and the increased accumulation of peptide β-CN(f193-206).

Several other unique peptides accumulated in the absence of PepNactivity. For the hydrolysis reactions of α_(S1)-CN(f1-9), thesepeptides corresponded to a decreased ability to liberate Lys³ fromα_(S1)-CN(f3-9) (FIG. 1). For the hydrolysis of β-CN(f193-209), thesepeptides corresponded to a decreased ability to liberate Tyr¹⁹³, Val¹⁹⁷,and Leu198 from derived peptides. The results for liberation of Lys andLeu residues are consistent with the reported AA specificities of PepNfor AA-ρNA substrates (Christensen et al., 1999). The activitiesmeasured for liberation of Tyr and Val from dipeptide substrates isroutinely reported to be relatively low (Niven et al., 1995). However,an evaluation of activity of purified lactococcal PepN for a trypticdigest of β-CN also indicated the ability of this peptidase to liberateTyr from β-CN(f193-202) and Val from β-CN(f170-176), as well as peptidescontaining Glu at the N-terminus (Tan et al., 1993).

The absence of PepX activity from CFE resulted in the accumulation ofα_(S1)-CN(f1-5) and α_(S1)-CN(f4-7), both peptides having an Xaa-ProN-terminus (FIG. 1). This is also consistent with the known substratespecificity of PepX (Christensen, 1999).

Surprisingly, no differences were detected for hydrolysis of either ofthe peptides with CFE from the endopeptidase deletion mutants, JLS232(ΔpepO) and JLS233 (ΔpepE), relative to wild type. The chromatograms forhydrolysis reactions of α_(S1)-CN(f1-9) by all six strains wereindistinguishable with respect to the rate of hydrolysis ofα_(S1)-CN(f1-9) and the accumulation of the primary product,α_(S1)-CN(f1-7), indicating that none of the deleted peptidases isresponsible for the hydrolysis of the Lys⁷-His⁸ peptide bond. Inaddition, the fractions analyzed from hydrolysis reactions ofβ-CN(f193-209) by all six strains contained several peptides with Pro²⁰⁴or Pro²⁰⁶ at the C-terminus (FIG. 1). The hydrolysis results indicatethat PepE and PepO either 1) do not have significant specificity forα_(S1)-CN(f1-9) or β-CN(f193-209) or derived peptides, 2) are notexpressed significantly under the growth conditions used, or 3) thepeptides that are substrates for these enzymes did not accumulate to asufficient level to be detected in our investigation. Since the deletionof PepE and PepO did not affect the rate of formation of these peptides,these results indicated the hydrolysis was due to an as yet unidentifiedendopeptidase and/or carboxypeptidase.

D. Hydrolysis of the Carboxy-terminal Protected Peptideβ-CN(f193-209-n-butyl amide)

To determine whether the carboxy-terminal hydrolysis of β-CN(f193-209)resulted from endopeptidase activity, hydrolysis products of thecarboxyl protected substrate β-CN(f193-209-n-butyl amide) wereidentified. Synthesis and purification of β-CN(f193-209-n-butyl amide)was done at the University of Wisconsin Biotechnology Center. In orderto reduce the extent of hydrolysis of peptides from aminopeptidaseactivity, hydrolysis reactions with β-CN(f193-209) andβ-CN(f193-209-n-butyl amide) were performed with CFE prepared fromJLS242 (ΔpepN). The predominant peptide formed from hydrolysis of bothβ-CN(f193-209) and β-CN(f193-209-n-butyl amide) was identified asβ-CN(f193-206), indicating the role of an endopeptidase in formation ofpeptides with Pro²⁰⁶ at the C-terminus.

E. Hydrolysis of Peptides at Low pH/High Ionic Strength

To evaluate hydrolysis of peptides under the pH and ionic conditionsassociated with ripening Cheddar cheese, reactions of L. helveticus wildtype CFE with α_(S1)-CN(f1-9) or β-CN(f193-209) were performed at pH 5.1in 120 mM MES buffer/0.68 M NaCl (4% NaCl).

Hydrolysis reactions with CFE were also performed at pH 6.5 (asdescribed above) for direct chromatographic comparison. The CFE werepreincubated (5 min) in the designated buffers before substrateaddition. Following addition of substrate, samples were incubated for20, 40 and 60 min in order to evaluate hydrolysis over time. Acomparative evaluation of chromatograms for each reaction condition (pH6.5 vs. pH 5.1/0.68 M NaCl) for α_(S1)-CN(f1-9) at a given reaction timerevealed no differences in the hydrolysis of the initial substrate andaccumulation of the primary hydrolysis product, α_(S1)-CN(f1-7). Insubtle contrast, the chromatograms for reactions with β-CN(f193-209)indicate a slight increase in the rate of hydrolysis of the initialsubstrate, but no significant difference in the subsequent hydrolysis ofthe primary product, β-CN(f194-209). The activity of an unidentifiedendopeptidase at pH 5.1 and 0.68 M NaCl indicates this enzyme may beimportant for initiating the hydrolysis of α_(S1)-CN(f1-9),β-CN(f193-209), and other Pro containing peptides.

Example 2 Identification and Characterization of PepO2

A. Materials and Methods

1. Bacterial Strains, Plasmid and Media

Lb. helveticus CNRZ32 (Khalid and Marth, 1990) and its derivatives weregrown in MRS broth (Difco Laboratories, Detroit, Mich.; DeMan et al.,1960) at 37° C. Lc. lactis LM0230 was propagated at 30° C. inM17-glucose broth (Difco Laboratories; Terzaghi and Sandine, 1975).Escherichia coli DH5α (Gibco-BRL Life Technologies Inc., Gaithersburg,Md., USA) and derivatives were grown in LB broth (Sambrook et al., 2001)at 37° C. with aeration. Agar plates were prepared by adding 1.5%(wt/vol) granulated agar (Difco Laboratories) to liquid media.Erythromycin (Sigma Chemical Co., St. Louis, Mo) at 500 μg/ml was addedto liquid media or agar plates to select for pJDC9 (Chen and Morrison,1988) in E. coli.

2. Screening of Lb. helveticus CNRZ32 Genomic Library

A previously constructed genomic library of Lb. helveticus CNRZ32 in E.coli DH5α (Nowakowski et al., 1993) was screened for endopeptidaseactivity using an amino-terminal blocked chromogenic substrate,N-acetyl-β-CN(f203-209)-ρ-nitroanilide (NA) (SynPep Co., Dublin,Calif.); this substrate is based on the C-terminal amino acid sequenceof Bos taurus β-CN. Pooled cultures (10 isolates/pool) were grownovernight in LB broth containing erythromycin. Cells were pelleted at13,000×g for 1 min at room temperature, washed and suspended in 10 mMBis-Tris, pH 6.5 (Sigma). Cell-free extracts (CFEs) from E. coli wereobtained by vortexing the samples with glass beads, alternating withcooling on ice, 1 minute each, repeated 2 times, and cell debris wasremoved by centrifugation for 1 min at 13,000×g. CFEs obtained frommid-log cultures of Lb. helveticus CNRZ32 and E. coli DH5α (pJDC9) wereused as positive and negative controls, respectively. The presence orabsence of endopeptidase activity was determined by adding 100 μl of CFEto 395 μl of 10 mM Bis-Tris (pH 6.5) containing 1 mMN-acetyl-β-CN(f203-209)-ρNA. The appearance of an intense yellow color(resulting from release of ρNA) within 15 min was a positive indicationof endopeptidase activity. In the coupled-reaction, 20 μl of CFE of E.coli DH5α containing aminopeptidase N activity (pJDC9::pepN) was used.All assays were conducted in duplicate.

3. Plasmid Isolation and Cloning

Plasmid isolation from E. coli was performed as described by Sambrook etal. (2001). Restriction enzymes and T4 DNA ligase were purchased fromGibco-BRL and used as recommended by the manufacturer. Electroporationof E. coli was performed using a Gene Pulser (Bio-Rad Laboratories,Richmond, Calif.) as recommended by the manufacturer.

4. DNA Sequencing and Sequence Analysis

All primers were synthesized by GIBCO-BRL Custom Primers (Grand Island,N.Y.). Polymerase chain reaction (PCR) and DNA sequencing reactions wereperformed in a Perkin-Elmer model 480 thermal cycler (The Perkin-ElmerCorp., Norwalk, Conn.). DNA sequencing reactions were conducted usingthe Prism™ Ready Reaction DyeDeoxy™ Terminator Cycle Sequencing Kit(Applied Biosystems, Inc., Foster City, Calif.). DNA templates werepurified with a Qiagen Inc. (Hilden, Germany) PCR purification kit.Sequencing was initially performed with M13/M13R primers (GIBCO-BRL). Asthe known sequenced progressed, new primers were designed accordingly.Additional primers were designed using the Affinity program supplied byRansom Hill Bioscience, Inc. (Ramona, Calif.). DNA sequencedetermination was conducted by the Nucleic Acid and Protein Facility ofthe University of Wisconsin-Madison Biotechnology Center, using an ABImodel 370/3 automated sequencer. Sequences were analyzed using the GCGsequence analysis package (Genetics Computer Group, Inc., Madison,Wis.). Protein homology searches were performed using the BLAST networkservice (Altschul et al., 1990). All reported DNA sequence data wasconfirmed by sequencing both DNA strands from at least two independentPCR products.

5. mRNA Analysis

Transcription of the PepO2 gene was investigated utilizing an 810-bpinternal PepO2 fragment (nucleotide 607 to 1416) amplified and the PCRproduct and labeled with digoxigenin (Genius™ system, BoehringerMannheim GmbH, Mannheim, Germany) for Northern hybridization. Theprimers used for probe amplification were YC-2290 (5'GATGCGATTGCACTCG)(SEQ ID NO:25) and YC-2000 (5'GATAGCGGCAGGGAAG) (SEQ ID NO:26). TotalRNA was isolated using the RNeasy™ kit (Qiagen). RNA molecular weightmarkers, solutions and reagents used in Northern hybridization andchemiluminescent detection were purchased from Boehringer Mannheim.Northern hybridizations were performed following the procedure suppliedby the manufacturer. Mapping of the 5′ end of the PepO2 transcript wasaccomplished using 5′ end rapid amplification of cDNA (5′RACE) kit(version 2.0; GIBCO-BRL). The gene specific primers used for 5′RACE wereYC-2340 (5′GTTTTCGGTTTGCTTTTG) (SEQ ID NO:27), YC-2600(5′CGGCATCTCTTTTGGC) (SEQ ID NO:28), and YC-2840 (5′GGACGATCGGCAGGG)(SEQ ID NO:29). First-strand cDNA synthesis was performed with primerYC-2340. Nested amplification of first-strand cDNA was carried out withprimer YC-2600 and the anchor primer supplied with the 5′RACE kit.Sequencing reactions were conducted with primer YC-2840 using the nestedamplification product as the template.

6. Synthesis of Peptide Substrates

The peptides α_(S1)-CN(f1-9) and β-CN(f193-209) were synthesized at theUtah State University Biotechnology Center. The synthesized peptideswere subsequently purified by collection of appropriate fractions frompreparatory RP-HPLC. The peptides were analyzed by mass spectrometry(MS) and confirmed by Edman degradation in an Applied Biosystems Model477B protein sequencer (Foster City, Calif.). The peptides werelyophilized and stored at −80° C. Stock solutions were prepared insterile ddH₂O and also stored at −80° C.

7. Peptide Hydrolysis Reactions

Peptide hydrolysis reactions were performed essentially as described inExample 1. A 10 μl aliquot of CFE (0.95-1.05 mg/ml protein) was dilutedin a total reaction volume of 500 μl of 0.1 M Bis-Tris buffer (pH 6.5).The reactions were initiated by the addition of the substrate and wereincubated at 37° C. for a minimum of 30 minutes. Initial substrateconcentrations in the reaction samples were 0.2 μg/μl for bothβ-CN(f193-209) and α_(s1)-CN(f1-9). Reactions were stopped byimmediately freezing at −20° C.

8. Peptide Separation and Identification

Samples were injected onto a 20 μl loop using a Gilson Model 231 sampleinjector equipped with a Model 401 dilutor module containing a 1:1ddH₂O:MeCN wash (Gilson Medical Electronics, France). The peptides wereseparated using a 250×2 mm Phenomenex Columbus C18 column (5μ, 100 Å)(Torrance, Calif.) preceded by a Brownlee RP-18 precolumn. The mobilephase flow rate and gradient was controlled with a Hitachi L-6200A pump(Hitachi Instruments, San Jose, Calif.). Mobile phases were continuouslydegassed by a slow helium sparge. Peptides were detected with a HitachiL-4500A diode-array detector in low absorbance mode. Data was collectedusing the Hitachi Chromatography Data Station Software with a wavelengthrange of 200-300 nm with a 4 nm spectral bandwidth and 3200 msecspectral interval.

Mobile phase A (MP-A) consisted of ddH₂O:MeCN (99:1) with 0.1% TFA andmobile phase B (MP-B) was ddH₂O:MeCN (20:80) with 0.05% TFA. Theseparation and elution of α_(S1)-CN(f1-9) hydrolysis samples wasaccomplished with the following gradient: 1-16% MP-B over 0-20 min at0.25 ml/min, 90% MP-B at 20-22 min at 0.25 ml/min, 90%-1% over 22-25 minat 0.25 ml/min. The separation and elution of β-CN(f193-209) hydrolysissamples was accomplished with the following gradient with respect toMP-B: 4-60% over 0-40 min at 0.25 ml/min, 60-98% over 40-41 min at 0.25ml/min, 98% from 41-45 min at 0.25-0.50 ml/min, 98%-4% over 45-47 min at0.50-0.25 ml/min. The pump back pressure was ˜1400 psi at T₀ andremained below 1600 psi for the duration of the gradients. Samples beingseparated for fraction collection were monitored in real time. Fractionswere collected manually taking into account a predetermined time for thepeptide to travel from the detector flow cell to the capture point.

The mass of RP-HPLC separated peptide fractions were determined using atriple quadrupole mass spectrometer (Micromass Quattro II) with anelectrospray ionization sources at the Utah State UniversityBiotechnology Center. To identify the hydrolysis products, the masseswere compared to calculated molecular weight of peptides and/or aminoacids derived from β-CN(f193-209) and α_(s1)-CN(f1-9).

9. Nucleotide Sequence Accession Number

The sequence for PepO2 has been submitted to GenBank and assignedaccession no. AF321529 (SEQ ID NOs:1 and 2).

B. Results

1. Screening of the Genomic Library

Prior to screening the Lb. helveticus genomic library, a number ofpreliminary tests were conducted. CFEs of Lb. helveticus CNRZ32 and E.coli DH5α were examined for endopeptidase activities capable ofhydrolyzing Ac-β-CN(f203-209)-ρNA. CFE from Lb. helveticus CNRZ32wild-type resulted in an intense yellow color (Abs410>0.30) within 15min, while E. coli DH5α CFEs resulted in only a very light yellow color(Abs410<0.025) after 10 h. To determine if any of the previouslyidentified Lb. helveticus proteolytic enzymes were required forhydrolysis of β-CN(f203-209), CFEs prepared from several peptidasemutants were examined for their ability to hydrolyzeAc-β-CN(f203-209)-ρNA (Table 2). Aminopeptidase N (PepN) was found to berequired for the release of ρNA from Ac-β-CN(f203-209)-ρNA. However, nohydrolysis of Ac-β-CN(f203-209)-ρNA was observed by CFEs prepared fromE. coli DH5α expressing Lb. helveticus PepN (JLS242; Christensen 2000).Together, these results indicate that PepN is required, but notsufficient, for ρNA release from Ac-β-CN(f203-209)-ρNA. Therefore, thegenomic library screen was performed using a coupled enzyme reactionwith PepN.

A genomic library of Lb. helveticus CNRZ32 in E. coli DH5α was screenedfor endopeptidase activities with Ac-β-CN(f203-209)-ρNA. Two isolates ofthe 1880 isolates screened had activity in a coupled reaction with PepN.Restriction endonuclease profiles of the two isolates were visuallyindistinguishable. One plasmid, designated pSUW99, was selected forfurther analysis.

TABLE 2 Ability of Lactobacillus helveticus CNRZ32 and its peptidase-deficient derivatives to hydrolyze N-acetyl-β-casein(f203-209)-ρ-nitroanilide Strains Relevant features Activity^(a) Reference CNRZ32Wild type + Khalid et al., 1990 JLS232 pepO⁻ derivative of CNRZ32 + Chenet al., 1998 JLS233 pepE⁻ derivative of CNRZ32 + unpublished JLS251prtH⁻ derivative of CNRZ32 + Pederson et al., 1999 JLS242 pepN⁻derivative of CNRZ32 − Example 1 JLS241 pepC⁻ derivative of CNRZ32 +Example 1 JLS243 pepX⁻ derivative of CNRZ32 + Example 1 ^(a)Enzymeactivity was determined with 1.0 mM of substrate at 37° C. for 30 min. Areaction was considered positive (+) if an absorbance of more than 0.025at 410 nm was observed.

2. Sequencing of the Endopeptidase Clone

Restriction mapping of pSUW99 revealed a 6.0-kb insert. Two 3.0-kb SstIfragments and two PstI fragments of 2.0-kb and 4.0-kb were obtained whendigested with restriction endonucleases SstI and PstI, respectively.Subclones containing individual SstI fragments or PstI fragments inpJDC9 were examined for endopeptidase activity withAc-β-CN(f203-209)-ρNA. Activity was only detected in strains containingone of the 3.0-kb SstI fragments, suggesting that the gene was presenton this SstI fragment and contained a PstI site.

The complete nucleotide sequence of the 3.0-kb SstI fragment encodingendopeptidase activity was determined (SEQ ID NO:1), and an open readingframe (ORF) of 1947-bp identified (SEQ ID NO:2). This ORF could encode apolypeptide of 649 amino acids with deduced mass of 71.4 kDa. Proteinsequence homology searches using current BLAST databases revealed highamino acid sequence similarity between the deduced amino acid sequenceand other LAB PepO-type endopeptidases (Chavagnat et al., 2000;Froeliger et al., 1999; Hellendoorn et al., 1999; Mierau et al., 1993;Tynkkynen et al., 1993). This protein has 56% identity and 72%similarity to Lb. helveticus CNRZ32 endopeptidase PepO (Chen and Steele,1998); therefore, this gene was designated PepO2. The Lb. helveticusPepO2 has 42% identity and 59% similarity to Lc. LactisPepO (Mierau etal., 1993; Tynkkynen et al., 1993), 41% identity and 61% similarity toLc. lactis PepO2 (Hellendoom et al., 1999), 38% identity and 57%similarity to the Streptococcus therinophilus PepO (Chavagnat et al.,2000), and 36% identity and 53% similarity to the Lb. rhamnosus PepO(Christensson et al., 2002). Significant similarity to mammalianmetallopeptidases, including endothelin-converting enzyme (45%similarity) and enkephalinase (neutral endopeptidase, NEP; 43%similarity) was also observed. The sequence motif His-Glu -Xxx-Xxx-His(SEQ ID NO:34), characteristic of many zinc-dependent metalloproteaseswas also identified in PepO2 between residues 497 and 501 (HEISH (SEQ IDNO:35)). The start codon of the ORF is preceded by a putative ribosomebinding site (AAGGAG; nucleotides −8 to −13) and putative promoter −10(TATGAT; nucleotides −32 to −37) and −35 (TTTTCA; nucleotides −56 to−61) sequences (Shine and Dalgarno, 1974). An inverted repeat(nucleotides 1967 to 1979 and 2000 to 2012) was observed in the 3′noncoding region and may function as rho-independent transcriptionalterminator with a ΔG [25°C.]=−21 kcal (Tinoco et al., 1973). No signalsequence was detected using a hydrophilicity plot constructed asdescribed by Kyte and Doolittle (1982).

3. mRNA Analysis

Northern hybridization using total RNA from an exponential culture ofLb. helveticus CNRZ32 resulted in the detection of a transcript with asize of 2.1-kb (data not shown). This size corresponds to the size ofthe PepO2 ORF, and indicates that PepO2 is monocistronic. Thetranscriptional start site for PepO2 promoter was mapped 26-bp upstreamof the PepO2 start codon by 5′ RACE.

4. Substrate Specificity of PepO2

To determine if PepO2 substrate specificity is similar to that ofpreviously described endopeptidases from Lb. helveticus CNRZ32, theability of CFE from E. coli DH5α expressing PepO2 to hydrolyzeN-benzoyl-Phe-Val-Arg-ρNA, N-benzoyl-Pro-Phe-Arg-ρNA andN-benzoyl-Val-Gly-Arg-ρNA was examined. These substrates had beenutilized previously to identify and differentiate PepO and PepE in agenomic library of Lb. helveticus constructed in E. coli DH5α (Chen andSteele, 1998; Fenster et al., 1997). Derivatives of E. coli DH5αexpressing PepO hydrolyzed N-benzoyl-Pro-Phe-Arg-ρNA andN-benzoyl-Val-Gly-Arg-ρNA, while derivatives of E. coli DH5α expressingPepE hydrolyzed N-benzoyl-Phe-Val-Arg-ρNA and N-benzoyl-Pro-Phe-Arg-ρNA.Hydrolysis of these substrates by PepO2, with or without PepN, was notobserved. Additionally, hydrolysis of Ac-β-CN(f203-209)-ρNA by CFEs ofE. coli DH5α Expressing Lb. helveticus PepO or PepE in coupled assayswith PepN was not observed. These results indicated that PepO2 substratespecificity is distinct from PepO and PepE. CFE from E. coli DH5αexpressing Lb. helveticus PepC or PepX were also examined in a coupledreaction with PepO2 (in place of PepN); the results indicated that onlythe combined activity of PepN and PepO2 was capable of releasing ρNAfrom Ac-β-CN(f 203-209)-ρNA.

To examine hydrolysis of the model casein-derived bitter peptides,β-CN(f193-209) and α_(s1)-CN(f1-9) by PepO2, RP-HPLC was performed toseparate and collect peptide hydrolysis products. No significanthydrolysis of either substrate was detected with CFEs from E. coli DH5α(pJDC9). However, significant hydrolysis of both β-CN(f193-209) andα_(s1)-CN(f1-9) was detected with CFEs from E. coli DH5α (pSUW99) (FIG.2). The predominant peptide fractions were collected and analyzed. PepO2was determined to hydrolyze β-CN(f193-209) at bonds Pro₁₉₆-Val₁₉₇,Pro₂₀₀-Val₂₀₁, and Pro₂₀₆-Ile₂₀₇. Hydrolysis of α_(s1)-CN(f1-9) wasobserved at Pro₅-Ile₆ bond (FIG. 3).

Example 3: Identification and Characterization of PepO3

A draft-quality genome sequence for L. helveticus CNRZ32 was obtainedand screened for genes encoding additional proteolytic enzymes. As isshown in Table 3, that effort revealed the CNRZ32 genome includes 8 ORFsencoding 3 known and 5 putative endopeptidases. The diversity ofendopeptidases in CNRZ32 is of considerable interest because theseenzymes play a key role in the hydrolysis of bitter peptides inbacterial-ripened cheeses, and high debittering activity is a knownattribute of this strain (Bartels et al., 1987). For this reason,endopeptidases were selected as the first targets for our functionalgenomics studies in CNRZ32. Since glycopeptides are not known to makeany contribution to bitter flavor defect in cheese, efforts weredirected toward the other 3 novel CNRZ32 endopeptidase genes: pepE2,pepF, and PepO3.

Lactobacillus helveticus CNRZ32 pepE2, pepF, and PepO3 genes wereisolated by PCR using primers designed from the genome sequence withadded Bam HI and Kpn I linkers. The amplicons were purified, cut witheach restriction endonuclease, then ligated into Bam HI and Kpn Idouble-digested pJDC9 (Chen and Morrison, 1988) and transformed intoEscherichia coli DH5a. Assays for endopeptidase activity intransformants. Cell-free extract (CFE) from E. coli transformantscontaining CNRZ32 pepE2, pepF, and PepO3 genes was prepared and assayedfor endopeptidase activity against chromogenic substrates in coupledassays with L. helveticus CNRZ32 PepN essentially as described by Chenet al. (2003). E. coli transformants containing CNRZ32 PepO2 (seeExample 2) were included as a positive control. Hydrolysis of bitterpeptides. E. coli CFE were incubated with the bitter peptides b-CN(f193-209) (1 mg/mL) or aS1-CN (1-9) (10 mg/mL) under simulated cheeseconditions (pH 5.0-5.2, 4% NaCl, 10° C.) as described in Examples 1 and2. Once again, E. coli transformants containing CNRZ32 PepO2 (seeExample 2) were included as a positive control. Reactions were stoppedby addition of equal volume of 10% TFA, then the remaining substrateconcentration was determined by RP-HPLC. Enzyme activity was expressedas a function of the change in substrate concentration over time.Finally, peptide products from β-CN (f193-209) or a 1-CN (1-9)hydrolysis were isolated by RP-HPLC and identified by mass spectrometryat the University of Wisconsin Biotechnology Center as described above.

Three putative genes in the CNRZ32 draft genomic sequence were studied.PepO3, pepF and pepE2 appeared to encode novel endopeptidases and thusmight contribute to debittering activity of this strain. BLAST searcheswith deduced products from these genes revealed strong amino acididentity to known endopeptidases from CNRZ32 and other LAB (Table 4). Asshown in Table 4, PepO3 and PepF activities were detected in E. colitransformants, but no activity was recorded in CFE from the culturetransformed with pepE2. CFEs from transformed E. coli were also testedfor the ability to hydrolyze, under simulated cheese ripening conditions(pH 5.0-5.2, 4% NaCl, 10° C.), the known bitter peptides β-CN (f193-209)and αS1-CN (f1-9).

As shown in FIG. 4, PepO3 and PepF each cleaved β-CN (f193-209), andPepO3 also hydrolyzed αS1-CN (f1-9). However, specific activitydeterminations showed both enzymes had significantly lower activitytoward these peptides than PepO2 (Table 5). No hydrolysis of eitherpeptide was detected after incubation with CFE from E. coli transformedwith pepE2. Identification of peptide products in reaction mixtures byRP-HPLC and mass spectrometry showed PepO3 was a post-prolineendopeptidase that hydrolyzed bonds Pro(206)-Ile(207),Pro(196)-Val(197), and Pro(200)-Val(201) in β-CN (f193-209) (FIG. 5). Asshown in FIG. 5, PepF also displayed post-proline specificity atPro(204)-Phe(205) and Pro(206)-Ile(207), but this enzyme also hydrolyzedβ-CN (f193-209) at the X-Gly bonds Lys(198)-Gly(199) andArg(202)-Gly(203). Specificity of PepO3 toward αS1-CN (f1-9) appears tobe limited to hydrolysis of the Pro(5)-Ile(6) bond.

L. helveticus CNRZ32 endopeptidase PepO3 is a functional paralog to thepost-prolyl endopeptidase PepO2. This enzyme displays a specificitytoward the known bitter peptides β-CN (f193-209) and αS1-CN (f1-9) thatis indistinguishable from that seen with PepO2. While the CNRZ32endopeptidase PepF also displayed post-prolyl specificity, this enzymewas also able to hydrolyze X-Gly bonds β-CN (f193-209), whereX=hydrophilic, charged residues.

TABLE 3 Lactobacillus helveticus CNRZ32 genes encoding known or putativeendopeptidases Gene Contig. Known (Reference) or Predicted Product pepE003-B Thiol-dependent endopeptidase (Fenster et al., 1997). pepE2 048PepE paralog; 53% identical to CNRZ32 endopeptidase PepE. pepF 104-APepF ortholog; 53% identical to Lactococcus lactis endopeptidase PepFpepO 026-C Endopeptidase O ortholog (Chen and Steele, 1998). PepO2 007-APost-prolyl endopeptidase (Chen et al., 2003). PepO3 077-A PepO/PepO2paralog; 62% identical to CNRZ32 endopeptidases PepO and PepO2 gcp 002-BGcp ortholog; 63% identical to predicted O-sialoglycoprotein ydiC 002-Bglycoprotein endopeptidase ortholog; 37% identical to predictedglycoprotein endopeptidase from Lactobacillus plantarum

TABLE 4 Substrate specificities of Lactobacillus helveticus CNRZ32endopeptidases cloned in Escherichia coli DH5a Substrate PepO2 PepO3PepF N-Benzoyl Pro-Phe-Arg-ρNA (w/o PepN) − − + N-BenzoylPro-Phe-Arg-ρNA + + + N-Benzoyl Phe-Val-Arg-ρNA + + + N-SuccinylAla-Ala-Pro-Phe-ρNA + + + N-Succinyl Ala-Ala-Ala-Val-Ala-ρNA + + +^(a)Coupled assays using E. coli CFE from transformants expressing Lb.helveticus CNRZ32 aminopeptidase PepN; + = OD410 > 0.030 within 15 minat 37° C. in HEPES (pH 7.0).

TABLE 5 Specific activity of Lactobacillus helveticus CNRZ32endopeptidases toward β-CN (f193-209) and α_(S1)-CN (f1-9)¹ SubstrateEnzyme β-CN (f193-209) α_(S1)-CN (f1-9) PepO2 2.9 × 102 (11) 1.9 × 103(57) PepO3 88 (2.7) 79 (8.6) PepF 14 (1.3) — None² 0.3 (0.2)  28 (6.6)¹nmoles substrate hydrolyzed/h/mg protein (±SE). Assays were performedusing CFE from E. coli DH5α transformed with CNRZ32 PepO2, PepO3, orpepF genes. ²Assay performed using CFE from untransformed E. coli DH5α.

Example 4 Continued Identification and Characterization of PepO3 andOther Endopeptidase Genes

A. Materials and Methods

1. Bacterial Strains, and Plasmid

Strains and plasmids used in this study are presented in Table 6. E.coli DH5α (Gibco-BRL Life Technologies Inc., Gaithersberg, Md.) andderivatives were grown in Luria-Bertani (Sambrook et al., 1989) mediumat 37° C. with aeration. Lc. lactis was grown at 30° C. without aerationin M17 (Difco, Detroit, Mich.) supplemented with 0.5% (w/v) glucose(G-M17), or lactose (L-M17). Lb. helveticus CNRZ32 was grown in MRSbroth (Difco, Detroit, Mich.; 12) at 37° C. without aeration. Agarplates were prepared by adding 1.5% (wt/vol) granulated agar (Difco,Detroit, Mich.) to liquid media with or without antibiotic. To selectfor E. coli strains carrying pBluescript II SK (+) (Stratagene, LaJolla, Calif.) and its derivatives, ampicillin (Sigma, St. Louis, Mo.)was added to media to a final concentration of 100 μg/ml. Erythromycin(Em; Sigma) was added to liquid media or agar plates to select forpJDC9, pTRKH2 and their derivatives in E. coli and Lc. lactis at 500μg/ml and 5 μg/ml, respectively. Bacteria were maintained as frozenstocks in liquid media containing 12% glycerol at −80° C.

2. Molecular Biology Techniques

DNA cloning and plasmid isolation techniques were performed according toSambrook et al. Restriction and modifying enzymes were used according tothe manufacturer's procedures (Invitrogen, Carlsbad, Calif.).Transformation of E. coli was performed with a Gene Pulser following themanufacturer's recommended instructions (Bio-Rad Laboratories, Richmond,Calif.). For transformation of Lc. lactis, the procedure of Holo and Nes(1989) was utilized. Lb. helveticus CNRZ32 chromosomal DNA was isolatedas described by Marmur (1961). Lactococcal plasmid DNA was isolated from50 mL culture using a modified alkaline lysis method (Sambrook et al.,1989) with an addition of lysozyme (30 mg/ml) to the resuspensionbuffer. For all large scale plasmid DNA extractions, final purificationof DNA was conducted using mini-columns from a QiaQuick PCR-Purificationkit (Qiagen, Valencia, Calif.), and DNA was dissolved in a final volumeof 30-50 μL. For isolation of DNA from gels, the Qiaquick Gel Extractionkit (Qiagen) was used.

3. DNA Amplification Via PCR

The DNA primers listed in Table 7 were synthesized by Invitrogen.Amplification reactions were typically performed using Taq DNApolymerase; for high fidelity reactions, Platinum Pfx DNA polymerase(Invitrogen) was utilized. The PCR cycling conditions for amplificationof DNA normally included 95° C. for 5 min, followed by 25-30 cycles of94° C. for 30 s, 50-60° C. for 30 s, and 72° C. for 1 min per kb of thefragment amplified followed by a single cycle of 72° C. for 7 min.“Direct Colony” PCR was used to screen transformants; the fragment ofinterest was amplified directly from the colonies without the initialDNA template isolation. A plastic sterile pipette tip was used to pickcolonies from plates and cells were mixed with 20 μL of standard PCRreaction mix containing Taq DNA polymerase (Invitrogen). To lyse thecells prior to standard cycling conditions, samples were heated to 98°C. for 10 min.

4. DNA Sequencing and Sequence Analysis

DNA sequencing was conducted with sequence-specific primers synthesizedby Invitrogen (Table 7). Sequencing reactions were performed using theABI Big Dye Reaction mix (Applied Biosystems, Foster City, Calif.), anda Perkin Elmer model 480 thermal cycler (Perkin Elmer Corp., Norwalk,Conn.). Sequence analysis was done on an ABI 377XL DNA sequencer by theNucleic Acid and Protein facility of the University of WisconsinBiotechnology Center (Madison, Wis.). The sequences were assembled andanalyzed using Lasergene (DNASTAR Inc., Madison, Wis.) sequence analysissoftware.

5. Cloning of Lb. helveticus CNRZ32 Endopeptidases

DNA and protein analyses of the draft sequence using online BLAST searchengines (on the World Wide Web at ncbi.nlm.nih.gov/BLAST/) detectedseveral new putative endopeptidases (Table 8). Lb. helveticus CNRZ32pepE2, pepF, and pepO3 genes were identified in the draft quality genomesequence of Lb. helveticus CNRZ32 (Table 8). Sequence specific primerswere designed (Table 7) with added Bam HI and Kpn I linkers to amplifyfragments including the ribosome binding site, promoter regions, codingsequence and inverted repeats present within 300-400 bp downstream ofthe coding sequence. The respective genes were amplified from the totalgenomic DNA of Lb. helveticus CNRZ32 using Platinum Pfx DNA polymerase(Invitrogen, Carlsbad, Calif.). The resulting amplicons were purified,digested with BamHI and KpnI and ligated to similarly double-digestedpJDC9 (Chen et al., 1987) and transformed into E. coli DH5α. Lb.helveticus pepE (Fenster et al., 1997) was amplified with its nativepromoter from the total DNA template of Lb. helveticus CNRZ32 usingPlatinum Pfx DNA polymerase and gene specific primers (Table 7) withSmaI and XbaI linkers. The resulting amplicon of ˜2.0 kb was digestedwith SmaI and XbaI and ligated to similarly digested pJDC9. Putativetransformants were screened using gene specific primers by Direct ColonyPCR. Restriction digest analysis and sequencing of the gene wereperformed to confirm the presence of the endopeptidase genes in pJDC9.Colonies that gave positive amplicons of expected size carried theclones of pepO3, pepF and pepE2 and pepE and were designated pSUW650,pSUW651, pSUW652 and pSUW653, respectively (Table 6). All clonedfragments were sequenced and found to be identical to the Lb. helveticusCNRZ32 genome sequence of the respective genes.

6. Construction of PpepO3 Translational Fusion Plasmids

The promoter region of pepO3 (GenBank accession number, AF019410, whichis hereby incorporated by reference) was amplified from pSUW650 usingPlatinum Pfx DNA polymerase (Invitrogen, Carlsbad, Calif.) and genespecific primers, KpnI-P_(pepO3)—For, BamHI-P_(pepO3)—Rev, (Table 2).The amplified promoter fragment of pepO3 was digested using BamHI andKpnI, and ligated to similarly digested pBluescript II SK(+)(Stratagene, La Jolla, Calif.) to generate translational fusion plasmidpSUW660. The ligation mixture was electroporated into E. coli DH5α, andblue-white screening by α-complementation using IPTG and X-Gal(Invitrogen, Carlsbad, Calif.) was employed to screen for putativetransformants. Direct Colony PCR using the above primers, restrictiondigest analysis and sequencing confirmed the presence of the promoterfragment of pepO3 in pSUW660.

7. Cloning of Lb. helveticus CNRZ32 Endopeptidases as P_(pepO3)Translational Fusions

The ORF of Lb. helveticus CNRZ32 pepO2 starting with the second codonand with transcription terminators was amplified from pSUWL29 (Table 6)via PCR using Platinum Pfx polymerase and primers, BamHI PepO2-ORF-Forand XbaI PepO2-ORF-Rev (Table 7). Similarly, the ORF of Lb. helveticusCNRZ32 pepE and pepF starting with the second codon and withtranscription terminators was amplified from genomic DNA of Lb.helveticus CNRZ32 via PCR using Platinum Pfx polymerase and primers,BamHI PepE-ORF-For and XbaI PepE-ORF-Rev; BamHI PepF-ORF-For and SacIPepF-ORF-Rev, respectively (Table 7). The ORFs of pepO2 and pepE weredigested with BamHI and XbaI and ligated to similarly digested pSUW660to generate pSUW661 and pSUW662, respectively (Table 6). The ORF of pepFwas digested with BamHI and SacI and ligated to similarly digestedpSUW660 to generate pSUW663. Ligation mixtures carrying pSUW661, pSUW662and pSUW663 were transformed into E. coli ABLE C (Stratagene, La Jolla,Calif.). Putative transformants of E. coli ABLE C carrying pSUW661,pSUW662, and pSUW663 were screened for presence of genes pepO2, pepE andpepF, respectively via Direct Colony PCR using gene specific primersthat amplified their respective ORFs. Positive endopeptidase activityconfirmed the expression of the peptidases in E. coli ABLE C.

8. Cloning of Lb. helveticus CNRZ32 Endopeptidases into Lc. lactis UsingpTRKH2

Lb. helveticus CNRZ32 pepO3 gene along with its promoter was cleavedfrom pSUW650 (Table 6) using BamHI and SacI sites and ligated tosimilarly digested pTRKH2 to generate pSUW664. Lb. helveticus CNRZ32P_(pepO3):pepO2 and P_(pepO3):pepE were cleaved from pSUW661 andpSUW662, respectively, using PvuII and XbaI sites and ligated to SmaIand XbaI digested pTRKH2 to generate pSUW665 and pSUW666, respectively.Lb. helveticus CNRZ32 P_(pepO3):pepF was cleaved from pSUW663 usingPvuII and SacI and ligated to SmaI and SacI digested pTRKH2 to generatepSUW667. Ligation mixtures were electroporated into Lc. lactis LM0230competent cells and transformants were isolated from LM17+Em platesafter 48 hours of anaerobic incubation. Putative transformants werescreened for the presence of constructs using Direct Colony PCR withprimers specific to the promoter of pepO3. However, several attempts toligate P_(pepO3):pepF to pTRKH2 followed by direct transformation intoLc. lactis LM0230 were unsuccessful. Positive endopeptidase activityconfirmed the expression of the peptidases PepO2, PepO3 and PepE in Lc.lactis LM0230.

9. Cell-free Extract (CFE) Preparation

Cultures were grown to late log phase (Abs_(600 nm) ˜2.0) and CFEs wereprepared in 50 mM 2-(N-Morpholino)ethanesulfonic acid (MES) buffer (5.0pH). Cells were broken by vortexing with 300 mg of glass beads for 3 minusing a Turbomix attachment to Vortex Gene2 (Scientific Industries, NY).CFEs from E. coli expressing CNRZ32 pepE, pepE2, pepF, and pepO3translational fusion genes were assayed for endopeptidase activityagainst chromogenic substrates as described by Chen et al. (1987).Protein concentration of the CFEs were determined using Protein AssayKit I from Bio-Rad and bovine serum albumin as the protein standard.

10. Preparation of CCS

CCS was prepared as described by Morris et al. (1988) using custom mademolds designed by Hassan. (2001). Briefly, 850 g of a three-week-oldCheddar cheese was grated and mixed with an equal quantity of sea sand.The sea sand-cheese mixture was placed in custom made molds and squeezedusing a Carver manual hydraulic press model no. 3912 (Fred S. Carver,Inc., Summit, N.J.). Pressure was gradually increased up to 10,000 psiand held at that pressure for ˜3 h. The CCS and cheese liquid fat werecollected and kept at 4° C. for 2 hours. This storage temperatureallowed the expressed fat to solidify as the upper layer, which wasremoved using a spatula. Residual fat was removed by centrifugation at1380×g for 10 minutes using an induction drive centrifuge model J2-21M(Beckman Coulter). Additionally, CCS was filtered using a 1.6 μm glassmicrofiber filter (Whatman International Ltd., England). The CCS wasfiltered sterilized by sequential passages through 0.45μ, and 0.22μcellulose nitrate filters (Nalgene Filtration Products, Rochester, N.Y.)and stored at −80° C. For peptide hydrolysis, CCS was prepared asdescribed above, boiled for 5 min at 100° C., extracted with equalvolumes of 100% ethyl ether, vacuum dried using a Savant Speed Vac (SC210A; Global Medical Instrumentation, Inc., Albertville, Minn.)resuspended in MES buffer, pH 5.0 (50 mM) buffer and concentrated 2×times to yield an effective reaction system.

11. Synthesis of Peptide Substrates

The peptides β-CN (f193-209), α_(S1)-CN (f1-9), α_(S1)-CN (f1-13), andα_(S1)-CN (f1-16), and α_(S1)-CN (f1-6) were synthesized at theUniversity of Wisconsin Biotechnology Center. The synthesized peptideswere subsequently purified by collection of appropriate fractions frompreparatory RP-HPLC. The peptides were analyzed by mass spectrometry(MS) using a Bruker Reflex II for matrix-assisted laserdesorption/ionization time of flight (MALDI-TOF). The peptides werelyophilized and stored at −80° C. Stock solutions were prepared insterile double distilled water and also stored at −80° C.

12. Peptide Hydrolysis Reactions

Peptide hydrolysis reactions with E. coli and Lc. lactis CFE wereperformed in 50 μL total volume. Each sample contained 10 μL of CFE(˜2.0 mg/mL protein), 10 μL of peptide and 30 μL of buffer. The bufferused for single peptide reaction and defined peptide mix was 120 mM MES(pH 5.1)/0.68 M NaCl (4% NaCl). CCS concentrate with a final pH of 5.2and the 4% salt concentration was used for the cheese model system. Thereactions were initiated by the addition of the substrate and thesamples were incubated at 10° C. for predetermined times. Initialsubstrate concentrations in the reaction samples were 1 mg/mL for β-CN(f193-209) and 10 mg/mL for α_(S1)-CN (f1-9). In the defined peptide mixreactions, peptides α_(S1)-CN (f1-13), and α_(S1)-CN (f1-16), andα_(S1)-CN (f1-6) were present at 5, 2.5 and 1 mg/ml concentrations,respectively. Reactions were stopped by addition of trifluoroacetic acid(TFA) to a 5% final concentration, samples were frozen at −20° C. untilanalysis.

13. Peptide Separation and Identification of Hydrolysis Products

Peptides were separated by RP-HPLC on a HP1100 series (AgilentTechnologies, Palo Alto, Calif.) system, using solvent A (0.1% TFA inHPLC grade water) and solvent B (0.085% TFA, 80% acetonitrile, 20% HPLCgrade water). The samples were analyzed on a AllTech Ultima C₁₈ column(250×2.1 mm, 5 micron particle size, 100 Å pore size; Alltech AssociatesInc., Deerfield, Ill.). The initial condition was 10% B, and a lineargradient from 10% to 60% was generated over 35 min for separation ofβ-CN (f193-209), the defined peptide mix and CCS samples. A lineargradient from 10-20% was generated over 15 min for separation ofα_(S1)-CN (f1-9) at a flow rate of 0.25 ml/min at 25° C. The elutedpeaks were detected by absorbance at 214 nm using a photodiode arraydetector spectrometer (HP1100 series). Before injection, samplesinactivated with TFA were thawed at room temperature and centrifuged(14,000×g for 5 min at 25° C.); 20 μl of the supernatant were directlyinjected into the column using a HP1100 series autosampler equipped witha dilutor module containing a water:acetonitrile wash solution (1:1;Agilent Technologies, Palo Alto, Calif.). Substrate concentration wasdetermined by peak area. Substrate was hydrolyzed to ˜10-20% andreaction rates were verified to be in the linear range when calculatingslope for specific activity determinations. Specific activity wascalculated as nmoles hr-1 mg-1 protein and reported values are correctedby subtracting the mean values obtained in the control treatments. Incase of α_(S1)-CN (f1-9) hydrolysis, average specific activity wascalculated since the initial lag phase was included in the calculation.

The masses of RP-HPLC separated peptide fractions were determined byusing a triple quadruple mass spectrometer (Micromass Quattro II,Micromass Ltd. Manchester, UK) with electrospray ionization sources atthe University of Wisconsin Biotechnology Center. To identify thehydrolysis products, the masses were compared to calculated molecularmasses of peptides and/or aminoacids derived from β-CN (f193-209) andα_(S1)-CN (f1-9). Sample preparation and identification of CCS peptidesusing tandem mass spectrometry was performed at the University ofWisconsin Biotechnology Center. Data dependent MS/MS switching on Q-TOFwas done using MassLynx software. Raw data was analyzed using MASCOT(Matrix Science Ltd., London, UK) and Spectrum Mill (AgilentTechnologies) software allowing for oxidized methionine and phosphotyrosine and serine modifications. SWISS PROT database search was usedfor peptide search.

14. Statistical Analysis

The rates of hydrolysis of the peptides were compared as a function oftime and calculated the specific activities in nmoles of peptidehydrolyzed per hour per mg protein. Results reported are mean values ±standard deviation of three independent trials, corrected by subtractingthe values obtained in the control treatments. Values are reported assignificantly different when a P-value of ≦0.05 was obtained usingStudent's t test. A single factor ANOVA was performed and the leastsignificant differences were calculated (SAS User's Guide: Statistics,1985) for separation of means for individual peptidases within singlepeptide peptide reactions, the defined mixture of cheese-derivedpeptides, and in CCS.

15. Nucleotide Sequence Accession Number

The nucleotide sequence of pepO3, pepF and pepE2 have been deposited inthe GenBank database under accession numbers AY355128, AY365129 andAY365130, respectively.

B. Results

1. Sequence Analysis

The ORF of pepO3 was 1929 bp encoding a polypeptide of 643 amino acidresidues with a deduced mass of 71.4 kDa (accession number AY355128).PepO3 was 62% identical to PepO2 and PepO in Lb. helveticus CNRZ32(accession numbers AF321529 and AF019410, respectively) and 78%identical to a hypothetical protein in Lb. gasseri (protein accessionnumber ZP_(—)00045894.1). The pepF ORF was 1794 bp encoding apolypeptide of 598 amino acid residues with a deduced mass of 66.4 kDa(AY365129). PepF has 52%, and 46% identity to previously characterizedPepF proteins from Lb. plantarum (protein accession numberNP_(—)785715.1) and Lc. lactis (accession number A55485), respectively.It has 75% identity to a hypothetical protein from Lb. gasseri (proteinaccession number ZP_(—)00046654.1). The pepE2 ORF was ¹³¹¹ bp encoding apolypeptide of 437 amino acid residues with a deduced mass of 48.5 kDa(accession number AY365130). PepE2 has 52% identity to previouslycharacterized PepE protein from Lb. helveticus CNRZ32 (accession numberAAB52540) and 80% identity to a hypothetical protein from Lb. gasseri(protein accession number ZP_(—)00047232.1). The ORFs of pepO3 and pepFcontain the sequence HEISH and HETGH, which is characteristic of theHEXXH-motif present in zinc metallopeptidases (Barret et al., 1998). Theamino acid residues important for substrate binding and catalysis bycysteine proteinases of prokaryotic and eukaryotic origin are conservedin PepE2 (Fenster et al., 1997).

2. Identification of Peptides from CCS

The peptide/protein summary of CCS generated by Spectrum Mill (AgilentTechnologies, Foster City, Calif.) and MASCOT (Matrix Science Ltd.,London UK) identified peptides and phosphopeptides mostly from α_(S1)-and β-CN. The majority of the peptides identified from β-CN were frompremature β-CN, β-CN (f60-81) and β-CN (f107-118). β-CN (f193-209) wasnot identified. The peptides identified from α_(S1)-CN were α_(S1)-CN(f1-9), α_(S1)-CN (f1-13), α_(S1)-CN (f1-16), α_(S1)-CN (f1-17),α_(s1)-CN (f24-41) and α_(S1)-CN (f24-39).

3. Peptide Hydrolysis and Specificity Using E. coli CFE

CFE of E. coli derivatives expressing PepO2, and PepO3 had significantlygreater activity than the control with both β-CN (f193-209) andα_(S1)-CN (f1-9), with highest activity detected with PepO2 (Table 9).PepF activity was detected with β-CN (f193-209) but not with α_(S1)-CN(f1-9). PepO, PepE and PepE2 activities were not observed with eitherpeptide.

The hydrolysis specificities of PepO2, PepO3, PepE and PepF with β-CN(f193-209) and α_(S1)-CN (f1-9), are presented in FIG. 6. PepO3 wasdetermined to be a post-proline endopeptidase, similar to PepO2, capableof hydrolyzing bonds Pro₍₂₀₆₎-Ile₍₂₀₇₎, Pro₍₁₉₆₎-Val₍₁₉₇₎, andPro₍₂₀₀₎-Val₍₂₀₁₎ and bonds Pro₍₅₎-Ile₍₆₎ of β-CN(f193-209) andα_(S1)-CN (f1-9), respectively (FIG. 6). PepF also hydrolyzedpost-proline bonds Pro₍₂₀₄₎-Phe₍₂₀₅₎ and Pro₍₂₀₆₎-Ile₍₂₀₇₎ butadditionally hydrolyzed X-Gly bonds, Lys₍₁₉₈₎-Gly₍₁₉₉₎, andArg₍₂₀₂₎-Gly₍₂₀₃₎ of β-CN(f193-209), and PepE hydrolyzed α_(S1)-CN(f1-9) at bonds Lys₍₃₎-His₍₄₎ and Lys₍₇₎-His₍₈₎.

4. Peptide Hydrolysis Using Lc. lactis CFE

The time course of hydrolysis for α_(S1)-CN (f1-9) in single peptide,defined peptide mix and CCS exhibited a slower initial rate (lag phase),which increased after 12 h of incubation until 48 hours with CFE ofLM0230 expressing PepO2, PepO3 and PepE (f1-9) (FIG. 7). A lag phase wasnot observed when lower concentrations (1 mg/ml) of α_(S1)-CN (f1-9)were used, suggesting that substrate inhibition maybe occurring athigher concentrations. LM0230 derivatives expressing PepO2 and PepO3under the control of the pepO3 promoter hydrolyzed both β-CN (f193-209)and α_(S1)-CN (f1-9) at a significantly greater rate than the controlsin all three systems (FIG. 7, Table 10). The peptide β-CN (f193-209) washydrolyzed completely within 12 hours when spiked CCS was incubated withCFE of strains expressing PepO2 or PepO3 (FIG. 8). The activity of PepO2with β-CN (f193-209) was 2-fold higher in CCS than alone or in thedefined peptide mix (Table 10). The activity of PepO2 with α_(S1)-CN(1-9) was reduced in CCS and the defined peptide mix, when compared toactivity observed on α_(S1)-CN (1-9) alone (Table 11).

LM0230 derivatives expressing PepE hydrolyzed α_(S1)-CN (f1-9) at asignificantly greater rate than control in single peptide and definedpeptide mix systems (FIG. 7, Table 11). In CCS, its activity wassignificantly inhibited although the expected hydrolysis products wereseen in the RP-HPLC chromatogram (FIG. 8, Table 11). There was alsohigher background activity detected in the CFE of control strain of Lc.lactis LM0230 for peptides α_(S1)-CN (f1-9) and β-CN (f193-209) in CCSthan in single peptide reactions or in the defined peptide mix; however,the peptide profile of control strain was similar to that observed attime zero (FIG. 8). Relative activity towards the other peptides in thedefined peptide mix was also calculated, with activity towards α_(S1)-CN(f1-9) taken as 100% (FIG. 9). Each peptidase hydrolyzed the differentpeptides at different rates. For e.g., amongst the α_(S1)-CN derivedpeptides, PepE had highest activity towards α_(S1)-CN (f1-9) andα_(S1)-CN (f1-6); PepO2 had highest activity towards peptides α_(S1)-CN(f1-9), and α_(S1)-CN (f1-13), and PepO3 had highest activity towardsα_(S1)-CN (f1-13) (FIG. 9).

TABLE 6 List of bacterial strains and plasmids Strain or plasmidRelevant characteristics Source or reference Escherichia coli DH5α E.coli strain with high Bethesda Research Lab efficiency cloning, enablesα-complementation ABLE C E. coli cloning strain Strategene, La Jolla, CAreduces copy number of ColE1 based vectors by 4 fold Lactobacillushelveticus CNRZ32 Wild type Laboratory strain Lactococcus lactis LM0230Plasmid free Efstathiou and Mckay, 1976 pTRKH2 Em^(r) lacZ; 6.9 kbO'Sullivan and Klaenhammer, 1993 pJDC9 Em^(r) lacZ; 6.85 kb Chen andMorrison, 1987 pTRKH-N 3.8 kb SmaI-SalI fragment Christensen, 1995containing Lb. helveticus CNRZ32 ligated into pTRKH2; Em^(r); 11 kb pepNpBlueskript II f1 origin in +/− orientation, Strategene, Inc. SK⁺Amp^(r) lacZ; 2.96 kb pSUWL29 3.0 kb PstI fragment Chen, 2001 containingCNRZ32 pepO2 ligated into pJDC9; ~9.0 kb pSUW650 2.6 kb BamHI-KpnI Thisstudy fragment containing pepO3 ligated into pJDC9 pSUW651 2.5 kbBamHI-KpnI This study fragment containing pepF ligated into pJDC9pSUW652 2.1 kb BamHI-KpnI This study fragment containing pepE2 ligatedinto pJDC9 pSUW653 2.0 kb SmaI-XbaI fragment This study containing pepEligated into pJDC9 pSUW660 0.2 kb KpnI-BamHI This study fragmentcontaining CNRZ32 P_(pepO3) ligated into pBlueskript II SK⁺ pSUW661 2.4kb BamHI-XbaI ORF This study containing CNRZ32 pepO2 ligated intopSUW660 pSUW662 1.7 kb BamHI-XbaI ORF This study containing CNRZ32 pepEligated into pSUW660 pSUW663 2.5 kb BamHI-SacI ORF This study containingCNRZ32 pepF ligated into pSUW660 pSUW664 2.6 kb PCR fragment This studycontaining CNRZ32 pepO3 from pSUW650 ligated into pTRKH2 pSUW665 2.6 kbPvuII-XbaI This study fragment containing CNRZ32 pepO2 from pSUW661ligated into pTRKH2 pSUW666 1.9 kb PvuII-XbaI This study fragmentcontaining CNRZ32 pepE from pSUW662 ligated into pTRKH2 pSUW667 2.9 kbPvuII-SacI fragment This study containing CNRZ32 pepF from pSUW663ligated into pTRKH2

TABLE 7 List of sequence specific primers¹. Descrip- tion or Primer namepurpose Sequence, 5′–3′ PepO3-For- forward; CGGGATCCTTTTGACTTTGGGTGAATBamHI amplifi- (SEQ ID NO:36) cation of pepO3 PepF-For- forward;CGGGATCCCTTAAGGGAGTTCGGAG BamHI amplifi- (SEQ ID NO:37) cation of pepFPepE2-For- forward; CGGGATCCTATAACAAGAACGCTAAGAA BamHI amplifi- (SEQ IDNO:38) cation of pepE2 PepE-For- forward; TCCCCCGGGATTAGATTAAGCAAG SmaIamplifi- (SEQ ID NO:39) cation of pepE PepO3-Rev- reverse;GGGGTACCACGAGAAGTGGTTAGTTGA KpnI amplifi- (SEQ ID NO:40) cation of pepO3PepF-Rev- reverse; GGGGTACCTTGGAGGAATTCATCTTTAG KpnI amplifi- (SEQ IDNO:41) cation of pepF PepE2-Rev- reverse; GGGGTACCCAGATAATGGCAAATGATAKpnI amplifi- (SEQ ID NO:42) cation of pepE2 PepE-Rev- reverse;GCTCTAGAGAAATTCGCCCTGGTC XbaI amplifi- (SEQ ID NO:43) cation of pepEKpnI forward; GGGGTACCGACTTTGGGTGAATC P_(pepO3)-For amplifi- (SEQ IDNO:44) cation of P_(pepO3) BamHI reverse; CGGGATCCCATTTTATTATTCAAAGAGAAP_(pepO3)-Rev amplifi- (SEQ ID NO:45) cation of P_(pepO3) BamHI PepF-forward; CGGGATCCCCAACAAGAAGCGAAGTC ORF-For amplifi- (SEQ ID NO:46)cation of pepF ORF SacI PepF- reverse; GCTGGAGCTCGTCAGCTTTTTGTATGGORF-Rev amplifi- (SEQ ID NO:47) cation of pepF ORF BamHI forward;CGCGGATCCAATTTAGCAAAAATC PepO2- amplifi- (SEQ ID NO:48) ORF-For cationof pepO2 ORF XbaI PepO2- reverse; GCTCTAGATCAATTATATAACTGATAC ORF-Revamplifi- (SEQ ID NO:49) cation of pepO2 ORF BamHI PepE- forward;CGGGATCCGAATTAACTGTGCAGG ORF-For amplifi- (SEQ ID NO:50) cation of pepEORF XbaI PepE- reverse; GCTCTAGAGAAATTCGCCCTGGTC ORF-Rev amplifi- (SEQID NO:51) cation of pepE ORF ¹The restriction sites flanking each primeris underlined and the name of the site is included in the primer name

TABLE 8 Lactobacillus helveticus CNRZ32 genes encoding known or putativeendopeptidases. Gene Known (reference) or predicted product pepEThiol-dependent endopeptidase pepE2 PepE paralog; 53% identical toCNRZ32 endopeptidase PepE pepF PepF ortholog; 53% identical toLactococcus lactis endopeptidase PepF pepO Endopeptidase O orthologpepO2 Post-prolyl endopeptidase pepO3 PepO/PepO2 paralog; 62% identicalto CNRZ32 endopeptidases PepO and PepO2 gcp Gcp ortholog; 63% identicalto predicted O-sialoglycoprotein endopeptidase Gcp from Lactobacillusplantarum ydiC glycoprotein endopeptidase ortholog; 37% identical topredicted glycoprotein endopeptidase from Lactobacillus plantarum

TABLE 9 Specific activity of cell free extract of Escherichia coli DH5αexpressing Lactobacillus helveticus CNRZ32 endopeptidases towardβ-casein (f193-209) and α_(S1)-casein (f1-9)¹. Strain α_(S1)-casein(f1-9) β-casein (f193-209) DH5α (pSUWL29) 3700 (19)^(a) 290 (19)^(a)DH5α (pSUW650)  85 (21)^(b)  88 (5.0)^(b) DH5α (pSUW51) N. D N. D DH5α(pSUW651) N. D  14 (3.0)^(c) DH5α (pSUW652) N. D N. D DH5α (pSUW653) N.D N. D ¹nmoles substrate hydrolyzed/h/mg protein (±SD). Values werecorrected by subtracting the mean values obtained in the controltreatments from CFE of Escherichia coli DH5α(pJDC9). Means (±SD) withdifferent letters are statistically different within a column at α ≦0.05. ²Assays were performed at cheese ripening conditions (pH 5.0-5.2,4% NaCl, 10° C.) using CFE from Escherichia coli DH5α (pJDC9)transformed with CNRZ32 pepO2 (pSUWL29), pepO3 (pSUW650), pepO (pSUW51),pepF (pSUW651) pepE2 (pSUW652), or pepE (pSUW653). N. D = Not detected

TABLE 10 Specific activity¹ of cell-free extract of Lactococcus lactisLM0230 expressing Lactobacillus helveticus CNRZ32 endopeptidases towardβ-casein (f193-209) as an individual peptide, in the defined peptidemix, and in Cheddar cheese serum. Single peptide Cheddar cheesePeptidase2 reaction Defined peptide mix serum PepO2 64 (1.5)^(a, B) 42(10)^(a, B) 120 (32)^(a, A) PepO3 81 (7.0)^(a, A) 40 (6.0)^(a, B)  61(23)^(b, A) PepE N. D N. D N. D ¹nmoles substrate hydrolyzed/h/mgprotein (±SD). Values were corrected by subtracting the mean valuesobtained in the control treatments. Means (±SD) with different lowercase letters within a colunm and with different upper case letterswithin a row, respectively, are statistically different at α ≦ 0.05. N.D = Not detected

TABLE 11 Specific activity¹ of cell-free extract of Lactococcus lactisLM0230 expressing Lactobacillus helveticus CNRZ32 endopeptidases towardα_(S1)-casein (f1-9) as an individual peptide, in the defined peptidemix and in Cheddar cheese serum. Individual peptide Defined peptideCheddar cheese Peptidase2 reaction mix serum PepO2 240 (10)^(a, A)  84(20)^(b, C) 150 (40)^(a, B) PepO3  41 (19)^(b, A)  38 (9.0)^(c, A)  63(25)^(b, A) PepE 190 (14)^(a, A) 120 (7)^(a, B)  31 (17)^(c, C) ¹nmolessubstrate hydrolyzed/h/mg protein (±SD). Values were corrected bysubtracting the mean values obtained in the control treatments. Means(±SD) with different lower case letters within a column and withdifferent upper case letters within a row, respectively, arestatistically different at α ≦ 0.05. ²Assays were performed under cheeseripening conditions, pH 5.0-5.2, 4% NaCl, at 10° C.; using cell freeextract from Lactococcus lactis LM0230 derivatives expressing CNRZ32PepO2, PepO3 and PepE.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references are specifically incorporated herein byreference.

-   U.S. Pat. No. 4,683,202-   U.S. Pat. No. 4,684,611-   U.S. Pat. No. 4,879,236-   U.S. Pat. No. 4,952,500-   U.S. Pat. No. 5,106,631-   U.S. Pat. No. 5,302,523-   U.S. Pat. No. 5,322,783-   U.S. Pat. No. 5,356,639-   U.S. Pat. No. 5,384,253-   U.S. Pat. No. 5,387,422-   U.S. Pat. No. 5,395,631-   U.S. Pat. No. 5,429,829-   U.S. Pat. No. 5,462,755-   U.S. Pat. No. 5,464,765-   U.S. Pat. No. 5,505,979-   U.S. Pat. No. 5,538,877-   U.S. Pat. No. 5,538,880-   U.S. Pat. No. 5,547,691-   U.S. Pat. No. 5,550,318-   U.S. Pat. No. 5,554,398-   U.S. Pat. No. 5,563,055-   U.S. Pat. No. 5,580,579-   U.S. Pat. No. 5,580,859-   U.S. Pat. No. 5,589,466-   U.S. Pat. No. 5,591,616-   U.S. Pat. No. 5,610,042-   U.S. Pat. No. 5,635,228-   U.S. Pat. No. 5,641,515-   U.S. Pat. No. 5,643,621-   U.S. Pat. No. 5,656,610-   U.S. Pat. No. 5,688,542-   U.S. Pat. No. 5,702,932-   U.S. Pat. No. 5,736,524-   U.S. Pat. No. 5,780,448-   U.S. Pat. No. 5,789,215-   U.S. Pat. No. 5,792,451-   U.S. Pat. No. 5,853,786-   U.S. Pat. No. 5,871,986-   U.S. Pat. No. 5,888,966-   U.S. Pat. No. 5,928,906-   U.S. Pat. No. 5,945,100-   U.S. Pat. No. 5,948,459-   U.S. Pat. No. 5,981,274-   U.S. Pat. No. 5,988,052-   U.S. Pat. No. 5,994,624-   U.S. Pat. No. 6,020,324-   U.S. Pat. No. 6,026,740-   U.S. Pat. No. 6,103,277-   U.S. Pat. No. 6,120,809-   U.S. Pat. No. 6,127,142-   U.S. Pat. No. 6,139,889-   U.S. Pat. No. 6,140,078-   U.S. Pat. No. 6,183,804-   U.S. Pat. No. 6,242,036-   U.S. Pat. No. 6,258,390-   U.S. Pat. No. 6,270,823-   U.S. Pat. No. 6,297,042-   U.S. Pat. No. 6,299,896-   U.S. Pat. No. 6,335,040-   U.S. Pat. No. 6,335,040-   U.S. Pat. No. 6,399,121-   U.S. Pat. No. 6,401,604-   U.S. Pat. No. 6,410,076-   U.S. Pat. No. 6,413,568-   U.S. Pat. No. 6,416,797-   U.S. Pat. No. 6,443,379-   U.S. Pat. No. 6,455,092-   U.S. Pat. No. 6,458,394-   U.S. Pat. No. 6,465,033-   U.S. Pat. No. 6,468,570-   U.S. Pat. No. 6,475,538-   U.S. Pat. No. 6,485,762-   U.S. Pat. No. 6,548,089-   U.S. Pat. No. 6,548,089-   U.S. Pat. No. 6,551,635-   U.S. Pat. No. 6,558,716-   U.S. Pat. No. 6,572,901-   Altschul et al., J. Mol. Biol., 215:403-410, 1990.-   Arora et al., Handbook of Fungal Biotechnology (Marcel Dekker),    2003.-   Ausubel et al., In: Current Protocols in Molecular Biology, John,    Wiley & Sons, Inc, New York, 1996.-   Bartels et al., Milchwissenschaft, 42:139-144, 1987b.-   Bartels et al., Milchwissenschaft, 42:83-88, 1987a.-   Benyx, Protein Expression Technologies: Current Status and Future    Trends (Horizon Bioscience), 2004.-   Broadbent et al., Appl. Environ. Microbiol., 68:1778-1785, 2002.-   Broadbent et al., J. Dairy Sci., 81:327-337, 1998.-   Carbonelli et al., FEMS Microbiol. Lett., 177(1):75-82, 1999.-   Chavagnat et al., FEMS Microbiol. Lett., 191:79-85, 2000.-   Chen and Morrison, Gene, 64:155-164, 1988.-   Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987.-   Chen and Steele, Appl. Environ. Microbiol., 64:3411-3415, 1998.-   Chen et al., Appl. Environ. Microbiol., 2002.-   Chen et al., Appl. Environ. Microbiol., 64:3411-3415, 1998.-   Chen et al., Appl. Environ. Microbiol., 69:1276-1282, 2003.-   Christensen et al., Antonie van Leeuwenhoek, 76:217-246, 1999.-   Christensen et al., Appl Environ Microbiol., 69(2):1283-1286, 2003.-   Christensen et al., Gene, 164:89-93, 1995b.-   Christensen et al., Intl. Dairy J, 5:367-369, 1995a.-   Christensen, In: Peptidases of Lactobacillus helveticus: role in    physiology and casein hydrolysis, University of Wisconsin-Madison,    2000.-   Christensson et al., Appl. Environ. Microbiol., 68:254-262, 2002.-   Cocea, Biotechniques, 23(5):814-816, 1997.-   DeMan et al., J. Appl. Bacteriol., 23:130-135, 1960.-   Detmers et al., Biochem., 37:16671-16679, 1998.-   Efstathiou and Mckay, J. Bacteriol., 130:257-265, 1976.-   Exterkate and Alting, Int. Dairy J., 5:15-28, 1995.-   Fechheimer et al., Proc. Natl. Acad. Sci. USA, 84:8463-8467, 1987.-   Fenster and Steele, J. Appl. Microbiol., 88(4):572-583, 2000.-   Fenster et al., J. Bacteriol., 179:2529-2533, 1997.-   Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348-3352, 1979.-   Froeliger et al., Infect. Immun., 67:5206-5214, 1999.-   Gomez et al., Milchwissenschaft, 51:315-319, 1996.-   Gopal, Mol. Cell. Biol., 5:1188-1190, 1985.-   Graham and Van Der Eb, Virology, 52:456-467, 1973.-   Harland and Weintraub, J. Cell Biol., 101(3):1094-1099, 1985.-   Hassan et al., Int. J. Food Microbiol., 64(1-2):199-203, 2001.-   Hellendoom et al., University of Groningen, Kerklaan 30, Haren 9751    NN, Netherlands, 1999.-   Holo and Nes, Appl. Environ. Microbiol., 55:3119-3123, 1989.-   Hwang et al. Crit. Rev. Ther. Drug Carrier Syst., 15(3):243-284,    1998.-   Inouye and Inouye, Nucleic Acids Res., 13:3101-3109, 1985.-   Johnson et al., In: Biotechnology And Pharmacy, Pezzuto et al.    (Eds.), Chapman and Hall, NY, 1993.-   Kaeppler et al., Plant Cell Reports, 9:415-418, 1990.-   Kaminogawa et al., J. Food Sci., 51:1253-1264, 1986.-   Kaneda et al., Science, 243:375-378, 1989.-   Kato et al, J. Biol. Chem., 266:3361-3364, 1991.-   Khalid and Marth, Appl. Environ. Microbiol., 56:381-388, 1990.-   Khalid et al., J. Dairy Sci., 74:29-45, 1991.-   Kok and De Vos, In: Genetics and biotechnology of lactic acid    bacteria, Glasson and de Vos (Eds.), Blackie Academic and    Professional, Glasgow, 1994.-   Kunji et al., Antonie van Leeuwenhoek, 70:187-221, 1996.-   Kyte and Doolittle, J. Mol. Biol., 157:105-132, 1982.-   Lee et al., J. Dairy Sci., 79:1521-1528, 1996.-   Lemieux and Simard, Lait., 71:599-636, 1991.-   Levenson et al., Hum. Gene Ther., 9(8):1233-1236, 1998.-   Madkor et al., J. Dairy Sci., 83:1684-1691, 2000.-   Mathiowitz et al., Nature, 386(6623):410-414, 1997.-   Mierau et al., J. Bacteriol., 175:2087-2096, 1993.-   Monnet et al., J. Biol. Chem., 269:32070-32073, 1994.-   Mulholland, In: Microbiology and biochemistry of cheese and    fermented milk, Law (Ed.), Blackie Academic and Profesiional,    Glasgow, 1997.-   Nardi et al., J. Bacteriol., 179:4164-4171, 1997.-   Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982.-   Nicolau et al., Methods Enzymol., 149:157-176, 1987.-   Niven et al., Appl. Microbiol. Biotech., 44:100-105, 1995.-   Nowakowski et al., Appl. Microbiol. Biotechnol., 39:204-210, 1993.-   O'Sullivan and Klaenhammer, Gene, 137:227-231, 1993.-   Omirulleh et al., Plant Mol. Biol., 21(3):415-28, 1993.-   Payne, J. Biol. Chem., 243:3395-3403, 1968.-   PCT Appl. WO 94/09699-   PCT Appl. WO 95/06128-   Pederson et al., J. Bacteriol., 181:4592-7, 1999.-   Perego et al., Mol. Microbiol., 5:173-85, 1991.-   Potrykus et al., Mol. Gen. Genet., 199:183-188, 1985.-   Pritchard and Coolbear, FEMS Microbiol. Rev., 12:179-206, 1993.-   Punt et al., Trends Biotechnol., 20(5):200-6, 2002.-   Rippe et al., Mol. Cell Biol., 10:689-695, 1990.-   Sambrook et al., Cold Spring Harbor Laboratory, old Spring Harbor,    N.Y., 2001.-   Sambrook et al., In: Molecular cloning, Cold Spring Harbor    Laboratory Press, Cold Spring Harbor, N.Y., 1989.-   SAS User's Guide: Statistics, Version 5^(th) Ed., SAS Inst. Inc.,    Cary, N.C., 1985.-   Schuppan et al., Science 297:2218-2220, 2002.-   Shine and Dalgamo, Proc. Natl. Acad. Sci. USA, 71:1342-1346, 1974.-   Smith et al., Anal. Biochem., 150:76-85, 1985.-   Tan et al., Appl. Environ. Microbiol., 59:1430-1436, 1993.-   Terzaghi and Sandine, Appl. Microbiol., 29:807-813, 1975.-   Tinoco et al., Nature (London) New Biol., 246:40-41, 1973.-   Tynkkynen et al., J. Bacteriol., 175:7523-7532, 1993.-   Vader et al., J Exp Med. 195(5):643-649, 2002.-   Wong et al., Gene, 10:87-94, 1980.

1. An isolated Endopeptidase polypeptide comprising an amino acidsequence with at least 90% identity to SEQ ID NO:4.
 2. The isolatedEndopeptidase polypeptide of claim 1, comprising an amino acid sequencewith at least 95% identity to SEQ ID NO:4.
 3. The isolated polypeptideof claim 2, comprising the amino acid sequence of SEQ ID NO:4.
 4. A foodadditive composition comprising an isolated PepO3 polypeptide comprisingthe amino acid sequence of SEQ ID NO:4, wherein the composition is aliquid, pellet, or powder.
 5. The food additive composition of claim 4,further comprising an isolated PepN polypeptide.
 6. The food additivecomposition of claim 4, further comprising an isolated PepO2 polypeptidehaving the amino acid sequence of SEQ ID NO:2.